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Process options
Lysis
Chemical
Thermal
Mechanical
Solids removal
Centrifugation
Filtration
Membrane separation
Flotation
Intermediate purification
Fractional precipitation
Membrane fractionation
Adsorption
High-resolution purification
Chromatography:
Ion-exchange
Reversed phase
Gel filtration
Affinity
Finishing
Drying
Formulation
Vialing
(b)
1.24 M 0
500 nm
trends in Biotechnology
Figure 1
Visualization of supercoiled plasmid DNA with atomic force microscopy: (a) a 6 kb
plasmid deposited onto mica functionalized with 3-aminopropyltriethoxy silane
(APTES, AP-mica) from TE buffer (20 mM Tris HCl, pH 7.6, 1 mM EDTA) with 100 mM
NaCl; (b) a high-resolution image of one molecule obtained by rescanning over a
500 3 500 nm area. (Images kindly provided by Dr Y. Lyubchenko.)
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13 kb
20 kb
80
29 kb
76 kb
100
60
SC DNA (%)
100
40
20
80
89 kb
60
40
40 kb
20
50 kb
76 kb
89 kb
0
0.0
0.5
1.0
1.5
2.0
Time of exposure to shear (sec)
0
0
5
10
15
20
Time of exposure to shear (sec)
trends in Biotechnology
Figure 2
Supercoiled plasmid-DNA content as a function of exposure to shear and plasmid size.
Clarified lysates containing plasmids of 13 kb (solid triangle), 20 kb (solid square) or
29 kb (solid circle) were subjected to shear rates of 1 3 106 s21 using a rotating disk
device operated at 27 700 rpm. Experimental conditions were as described in Ref. 24.
The curves in the inset are the predictions based on a first-order-kinetic model of damage to plasmid DNA; the data points in the inset were obtained by the authors for
clarified lysates containing plasmids of 76 kb (inverted solid triangle) or 89 kb (solid
diamond). Abbreviation: SC, supercoiled circular.
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100
80
60
40
20
0
1
2
3
Experimental condition
5
trends in Biotechnology
Figure 3
Supercoiled plasmid-DNA content after shearing in the absence or presence of air
liquid interfaces for a 20 kb plasmid under different DNA concentration and ionicstrength conditions: (1) clarified alkaline lysate (20 mg ml21); (2) ultrapure plasmid in
TE buffer (20 mg ml21); (3) ultrapure plasmid in TE buffer (5 mg ml21); (4) ultrapure
plasmid in TE buffer (0.2 mg ml21); (5) ultrapure plasmid in 150 mM NaCl, TE buffer
(0.2 mg ml21). The solutions were stirred for 5s at 27 700 rpm in the absence of
airliquid interfaces (filled bar) or at 26 650 rpm in the presence of airliquid interfaces
(open bar). Data shown are the average of two independent experiments, error bars
reflect standard deviation. Experimental conditions were as described in Ref. 24.
Abbreviations: TE buffer, 10 mM TrisCl, pH 8, 1 mM EDTA. (Adapted from Ref. 24.)
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50
Intact cells (%)
100
40
Viscosity (mPas)
10
1
0.1
30
0
10
20
30
Time (sec)
40
20
10
0
0
100
200
Time (sec)
300
400
trends in Biotechnology
Figure 4
Viscosity profile as a function of time after adding NaOH/sodium dodecyl sulphate
(SDS) to a suspension of Escherichia coli cells containing a 76 kb plasmid. The inset
shows the percentage of intact cells for the initial period of the reaction. The reaction was performed inside a co-axial cylinder viscometer operated at a shear rate of
231 s21. (Adapted from Ref. 26.)
Strain
100%, 0.2
Frequency
(Hz)
Phase
angle d
Storage
modulus G9
(Pa)
Loss
modulus G0
(Pa)
Dynamic
viscosity
(Pas)
Phase
angle d
Storage
modulus G9
(Pa)
Loss
modulus G0
(Pa)
Dynamic
viscosity
(Pas)
1
5
13.2
13.4
142.0
471.0
33.4
113.0
5.3
3.6
22.1
27.9
69.3
172.0
28.2
90.9
4.5
2.9
aTable
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100
100
80
90
60
80
40
70
20
0
60
0
5000
10000
Speed (rpm)
15000
trends in Biotechnology
Figure 5
Clarification and supercoiled plasmid-DNA content in supernatants as a function of
centrifugal force. A lysate containing a 20 kb plasmid was filtered through muslin cloth
before centrifugation in a continuous-flow tubular centrifuge. Clarification was monitored by optical density at 600 nm (OD), relative clarification was calculated by normalizing OD values to those obtained for the lysate centrifuged at 15 000 rpm. Supercoiled plasmid content was quantified in solution using a fluorescence-based method17.
Abbreviation: SC, supercoiled circular.
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(b)
(a)
M
CHR
OC + CHR
SC
CHR
trends in Biotechnology
Figure 6
Plasmid purification by retention of contaminant Escherichia coli chromosomal DNA on nitrocellulose: (a) agarose gel electrophoresis and
(b) Southern blot analysis using a sequence specific for the E. coli chromosome as a probe. Abbreviations: M, supercoiled DNA ladder
(216 kb); (1) plasmid DNA (500 ng) purified by isopropanol precipitation; (2) same as 1 followed by anion-exchange chromatography; (3)
same as 2 but filtered through nitrocellulose before anion-exchange chromatography; (4) material retained by the nitrocellulose membrane
(DNA amount loaded was not determined). Densitometric scanning of the Southern blot gave relative signal values of 1:0.24:0.07 for lanes
1, 2 and 3, respectively. Abbreviations: CHR, chromosomal DNA; OC, open circular plasmid DNA; SC, supercoiled circular plasmid DNA.
(Reprinted from Levy et al.39 with permission.)
There have been several reports on the use of fractional precipitation to purify plasmid DNA. Some of
these use divalent and trivalent metal salts and combinations of polyethylene glycol (PEG) and salts as well
as PEG alone40; there are also some earlier published
reports of PEG precipitation of DNA41,42. Chen and
Ruffner43 reported the use of 1 M magnesium chloride
and 3.3% (w/v) PEG to remove debris, chromosomal
DNA, RNA and protein followed by 1.5 M salt and 5%
(w/v) PEG to precipitate plasmid DNA. The basis of
selective precipitation of RNA and DNA might partly
be the differences in charge density between the plasmid DNA and other nucleic-acid contaminants and
partly the higher regional phosphate-charge density of
inter-helix junctions found in transfer RNAs and
recombinant RNAs compared with DNA44. Alternative approaches using ammonium sulphate45, spermidine46 and spermine46 fractional precipitation have also
been described.
High-resolution separation
It is unlikely that plasmid DNA of the quality required
for clinical purposes can be produced without the use
of chromatography; this has been the subject of several
studies13,14,47,48. In this article, the only element that will
be addressed in particular is the impact of the size of
the macromolecules and the way this affects available
chromatographic media. Plasmid DNA is large, relative
to the proteins for which most macromolecule-directed
chromatographic media were created; the effect of this
is illustrated in Fig. 7, which relates to experiments with
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6
10
5
9
4
3
8
6
2
7
5
1
0
34
1 2
0
20
40
60
80
100
120
Reciprocal of mean particle radius (mm1)
140
trends in Biotechnology
Figure 7
Binding capacity as a function of particle diameter of resin for a 6.9 kb plasmid binding to commercial anion-exchange adsorbents. Feedstock and column equilibration
conditions: feedstock, resuspended PEG precipitated material supplemented with
0.30.5 M NaCl/10 mM Tris-HCl pH 8; column equilibration 1 mM EDTA containing
0.30.5 M NaCl. Chromatographic media tested: (1) STREAMLINE DEAE (186 mm);
(2) DEAE Sepharose FF (93 mm); (3) Q Sepharose FF (93 mm); (4) DE52 Cellulose
(75 mm), the particle is a rod 120 mm length and 40 mm diameter; (5) Source 30Q
(30 mm); (6) Source 15 Q (15 mm); (7) Q Sepharose XL (93 mm); (8) Q Hyper D (M) 65
(65 mm); (9) Fractogel EMD DMAE 650 (M) (65 mm); and (10) POROS 50 DEAE (50 mm).
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10
SC
Sheared DNA
trends in Biotechnology
Figure 8
Effect of shear on plasmid-DNApolylysine complexes. The complexes were mixed at a charge ratio of 1.5 using a semi-automated syringe
pump and sheared using a rotating disk device operated at a shear rate of 1 3 106 s21 for 5s using the rotating disk device (Ref. 31). The
sheared complexes together with unsheared controls were treated with trypsin before agarose gel electrophoresis analysis. Lanes: (1,2)
unsheared and sheared plasmid (29 kb) in TE buffer; (3,4) unsheared and sheared plasmid in TE buffer/150 mM NaCl; (5, 6) unsheared and
sheared plasmid-polylysine (26 kDa) in TE buffer; (7,8) unsheared and sheared plasmid-polylysine (26 kDa) in TE buffer/150 mM NaCl; and
(9,10) unsheared and sheared plasmid-polylysine (100 kDa) in TE buffer. Abbreviations: SC, supercoiled circular plasmid DNA; TE buffer,
10 mM TrisCl, pH 8, 1mM EDTA. (Adapted from J.T. Tsai, PhD thesis, University of London, London, UK, 1999)
As mentioned previously, final-product-release specifications are likely to require .90% of the plasmid
DNA molecules to be in the supercoiled form14,67. These
forms are produced within the cell by the action of DNAsupercoiling enzymes, such as DNA topoisomerases,
which introduce negative superhelical twists into plasmid-DNA molecules. OKennedy et al.68 have shown
that the proportion of supercoiled forms can depend
strongly on the type of growth medium used. This
suggests that the proportion of supercoiled forms can
be modulated using different growth conditions and
could therefore be a possible target for recombinant
genetic modification. Supercoiled plasmids exhibit a
distribution with respect to the number of superhelical
twists that have been introduced, and this supercoiling
density can affect the transcription efficiency of plasmid-encoded genes. This is known to depend on factors
that include growth temperature and exposure to cold
or heat shocks69.
Conclusion
The biochemical engineering studies reviewed here
have created the foundation for the efficient isolation
of plasmid DNA. At present, scales and efficiencies are
modest; however, if DNA vaccines prove to be effective, the ultimate scale will be large. With tuberculosis,
often in forms that are resistant to drugs, causing three
million deaths per year worldwide, malaria (two
million deaths per year) and AIDS (one million deaths
per year), there is a great need for stable new vaccines;
an influenza epidemic could demand much-greater
quantities quickly3. Recent reports on the use of DNA
vaccines in animal studies are promising70, although the
situation with gene therapy is less clear at present.
However, the potential of gene therapy and DNA
vaccines to address currently incurable diseases indicates
a large possible demand. Given these two broad categories of potential use, the establishment of strong
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biochemical engineering foundations will be important as a general guide to future processing. As indicated
in this review, the interactions between particular stages
are especially pronounced, a whole bioprocess approach
will therefore be even more important for DNA than
for proteins.
Acknowledgments
We are very grateful to our colleagues (D. Cooke,
M. Hoare, D. Kendall, L. Lee, G. Lye, P. McHugh,
M. Tservistas and J. Ward) for valuable advice and
comments. We are also grateful for the support of
the Biotechnology and Biological Sciences Research
Council.
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