THE JOURNAL OF GENE MEDICINE
J Gene Med 2004; 6: S45–S53.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.511
Animal-free production of ccc-supercoiled plasmids
for research and clinical applications
Martin Schleef*
Torsten Schmidt
PlasmidFactory GmbH & Co. KG,
Meisenstr. 96, D-33607 Bielefeld,
Germany
*Correspondence to: Martin Schleef,
PlasmidFactory GmbH & Co. KG,
Meisenstr. 96, D-33607
Bielefeld, Germany. E-mail:
martin.schleef@plasmidfactory.com
Copyright  2004 John Wiley & Sons, Ltd.
REVIEW ARTICLE
Summary
The topological structure of plasmid DNA can be characterized by capillary
gel electrophoresis (CGE analysis) – an important tool for quality control
and stability assessments in DNA storage or application. Hence, a large-scale
manufacturing process was developed that allows the removal of undesired
open circular (oc) or linear plasmid topologies, bacterial genomic DNA, RNA,
proteins as well as lipopolysaccharides (endotoxins) and results in obtaining
supercoiled (covalently closed circular, ccc) plasmid DNA in a pure form
without using any animal-derived substances.
Using CGE, the development and in-line monitoring for pharmaceutical
plasmid production starting from fermentation control throughout the whole
manufacturing process including the formulated and filled product can
be performed the first time in a way conforming to good manufacturing
practices (GMP). Plasmid stability data were obtained from analysis of shear
effects influencing the plasmid quality in DNA drug delivery formulation and
application (e.g. gene gun or jet injection). The physical stability of plasmid
DNA is for the first time evaluated in DNA storage experiments on the level
of different plasmid forms. Copyright  2004 John Wiley & Sons, Ltd.
Keywords non-viral vectors; ccc-supercoiled plasmids; capillary gel elec-
trophoresis; large-scale production; gene therapy
Introduction
The production and quality control of plasmid DNA required for gene
therapy, DNA vaccination or virus production for research and therapeutic
use, as well as for other pharmaceutical delivery in gene medicine, is
heavily improved by novel technologies for separating different plasmid
topologies. A comprehensive overview on the state-of-the-art in plasmid DNA
pharmaceuticals was published earlier [1].
Gene therapy
Gene therapy is based on the finding that certain diseases are caused by genetic
defects. The addition of correct genetic information may compensate them.
Further approaches make use of the fact that additional genetic information
may have the potency to induce further therapeutic effects, like labelling
tumor cells by the application of therapeutic genes, which indicate target cells
to the immune system.
DNA vaccination
Vaccination with DNA (for review, see [1]) instructs an organism (e.g. in
parallel to human also mice, monkey, fish or pigs) how to react to a future
S46
challenge by a pathogen or parasite – it is preventive.
These vaccines should generate a sufficient immune
response for protecting the treated organisms. The vaccine
API (active pharmaceutical ingredient) should be produced
and applied in a safe manner, and should be applicable
even for the vaccination of populations.
Classic vaccines for human prevention are, e.g.,
life-attenuated viruses (e.g. polio or measles), inacti-
vated microorganisms (e.g. cholera bacteria), inactivated
viruses (e.g. influenza), recombinant vaccines (e.g. HBV
surface antigens), as well as pseudo-viral or virus particles
produced in yeast, hybrid vectors (bacterial or viral) or
synthetic peptides.
Novel DNA vaccines may consist of no protein
component at all or combinations of DNA, protein
and other substances responsible for, e.g., compacting
or targeting. The DNA encodes the antigen. The cell
transfected with such a DNA molecule or complex is in
consequence a vaccine-manufacturing unit by itself and
produces the ‘drug’ specifically for its own purpose. This
intracellular production process completely reflects the
genetic background of the cell, since it experiences the
gene expression like a real infection. In consequence, the
transcribed, translated, and maybe post-translationally
modified, Ag protein is nearly identical – different than
recombinant protein vaccines – to the Ag pattern of an
infection.
Gene delivery
The transfer of DNA has evolved from viral and liposomal
gene transfer technologies to naked and other compacting
or complexing and ideally targeting substances and is
gaining increasing importance for genetic immunization
and other gene therapy applications [2,3]. Various
in vitro and in vivo procedures have been employed
to deliver naked or complexed DNA into the desired
cells or tissues, like simple needle injection, particle
bombardment, in vivo electroporation or jet-injection
[4–7]. Non-viral techniques have numerous advantages,
such as circumvention of utilization of recombinant viral
particles, minimal or no immune response towards the
foreign DNA applied, and no toxicity.
The majority of non-viral gene transfer technologies
are used in the context of DNA-vaccination studies to
introduce DNA constructs, which encode proteins or
peptides capable of inducing immune responses, which
result in antibody production in the host. For these
intradermal or intramuscular applications, utilization of
naked DNA has proven to be an efficient DNA vaccine
against different viral infections or as cancer vaccine in
numerous animal models [8–11].
Wolff and co-workers were the first to show that simple
application of naked DNA is sufficient for gene transfer
leading to the expression of the transgene [12]. However,
although needle injection can transduce naked DNA into
muscle tissues, this technique was largely inefficient for
other tissues including tumors. Meanwhile, numerous
Copyright  2004 John Wiley & Sons, Ltd.
M. Schleef and T. Schmidt
studies have dealt with the optimization of this procedure
[13–16].
The particle bombardment or gene-gun technique
using DNA-coated gold or tungsten microparticles is
effective in gene transfer for different tissues. However,
for this ballistic gene transfer, DNA penetration is
limited and cannot reach deeper areas of the targeted
tissues. Therefore, most studies that use the particle
bombardment for non-viral gene transfer are aimed
at applications for vaccination or immune stimulatory
approaches targeting dermal or sub-dermal areas [17].
Among the various non-viral gene delivery technologies
jet-injection is gaining increasing acceptance, since this
technique allows gene transfer into different tissues with
deeper penetration of the applied naked DNA into the
targeted tissues. Meanwhile, this technology can achieve
comparable transfer efficiencies as seen, e.g., by particle
bombardment [7,18].
Plasmid vectors for clinical
application
Typically, vector plasmids are double-stranded circular
DNA molecules with a size between two and some
hundred kilo base pairs (kbp) [19,20]. The combinatory
biology allows the design of elements of a plasmid
DNA vaccine very precisely, like combining different
epitopes, generating functional domains of proteins in
diverse constellations and testing the constructs for their
function automatically [21]. In addition, the option
to synthesize sequences specifically is important, since
mRNA transcripts of these vectors may be stabilized [21]
and the vector safety may be increased. The presence of
sequences with unknown potency or adverse properties
may be avoided and the addition or modification of useful
sequences is available.
The modes of action of DNA vaccines or therapeutic
plasmids, as well as examples for their application, have
been discussed already [1]. Important developments were
recently observed in DNA drug formulation: production
of the key intermediate (plasmid DNA) and quality
assurance of such therapeutics. In particular, applications
for DNA vaccines in veterinary sciences are expected [22],
since animals have to be vaccinated against multiple
pathogens (viruses, parasites and other intracellular
pathogens), which in consequence may not be combined
in one dose. Costs for storage and application will
be significantly reduced. Based on this idea, certain
intelligent vector systems were developed with multiple
different expression elements on one plasmid molecule
[23] or vectors where diverse immunogenic epitopes are
shuffled [24,25]. However, such approaches require a
clearly adjusted quality assurance in manufacturing. The
different epitopes may be shuffled, but will not necessarily
be expressed in equimolar amounts. Alternatively,
different plasmid constructs may be produced individually
and mixed subsequently at defined amounts. The
J Gene Med 2004; 6: S45–S53.
Production of ccc-Supercoiled Plasmids
drawback is the lower dose per construct, limitations
in the total applicable DNA amount and increasing costs
for multiple independent production runs.
Production of ccc-supercoiled plasmid
DNA
Traditional methods of purifying plasmids usually require
sophisticated methodology, if the DNA has to be separated
from contaminating organic components. Already the
purification of plasmid DNA for research applications
requires high-quality materials and technologies to obtain
a reproducible and appropriate quality. Besides the use
of qualified production strains for the microbiological
amplification of the required plasmid DNA, the cultivation
of biomass in fully defined media has become a safety
issue with respect to the recent discussion on the
use of animal-derived raw materials (e.g. peptones,
extracts). Consequently, avoiding any of these materials
and also antibiotics in cultivation and other process
steps is a general pre-requisite for the application of
DNA vectors – already in research and development
(R&D) – and certainly in the clinics (see below).
The development of processes for plasmid DNA
manufacturing (for reviews, see [26,27]) is – compared
with process development, e.g., in classic vaccine
production – significantly shorter, since core elements of
the DNA process are nearly identical for each plasmid
of up to 10 kbp [28]. Recently, a requirement for
producing larger plasmids has been observed. This is
due to larger sequences within the vectors, which need
further modifications within the manufacturing process.
For manufacture of plasmid DNA drugs in a sufficient
quality, appropriate production processes are required,
which have to fulfil the requirements of respective
guidelines and laws. GMP (good manufacturing practices)
have to be applied in production and quality assurance.
These requested standards can only be obtained in specific
manufacturing and quality control facilities. The main
requirements in plasmid purification are:
1. Separation of plasmid DNA from any other sub-
stance deriving from the purification process or the
bacterial host cell (e.g. cell wall residues, lipopolysac-
charides – LPS, bacterial genomic DNA, RNA, proteins,
lipids, etc.),
2. Purification of the ccc form from all other DNA forms
to obtain a homogenous product, and
3. Protection of the desired supercoiled plasmid form
(ccc) throughout the manufacturing process.
The standard process in plasmid production starts with
the stable transformation of the plasmid vector into a
defined plasmid production Escherichia coli (E. coli) host
strain followed by cultivation in a bioreactor, alkaline
lysis of the bacterial biomass, filtration and several
chromatography steps to obtain the requirements listed
above (for review, see [29]).
Copyright  2004 John Wiley & Sons, Ltd.
S47
In bacterial cultivation for plasmid productions,
typically different plasmid forms appear. The ccc plasmid
topology is the most compact structure and is expected to
be the most active topology. If one strand is broken
(nicked), the oc form results. This is caused either
naturally by processes within the plasmid-producing
bacteria or while processing the biomass (enzymatic
or mechanic degradation). Linear forms are generated
if both strands are cleaved once at approximately the
same position. In addition, plasmids may appear as
oligomeric forms, e.g. concatemers, which may in turn
exist in different topologies (see Figure 1). The only intact
and undamaged form is the ccc-supercoiled DNA. Linear
forms as well as oc forms are damaged at different gene
locations randomly, which make these forms inefficient, if
promoter or gene coding regions have been destroyed.
Any process development has to be accompanied by
a powerful in-process-control (IPC) system to generate
data on the characteristics of the plasmid molecules. In
particular, methods are required for obtaining supercoiled
covalently closed circular (ccc) plasmid DNA in pure
form. Commonly, other plasmid topologies appear as well,
which have to be separated from the desired product.
A main focus in the development of pharmaceuti-
cal DNA medicines is the stability of DNA, which has
two major aspects: on the one hand, manufacturing,
formulation, storage and application should be easy
and cost-efficient. On the other, this substance must
be removed completely from the manufacturing envi-
ronments at product change. The aspect of changeover
is extremely important if different DNAs are produced
within the same facility.
The product stability in DNA manufacturing, formula-
tion, storage and application mainly reflects the plasmid
topology. For the first time, the characterization of this
three-dimensional structure was possible using capillary
gel electrophoresis (CGE) technology [30]; for review,
see [31]. The CGE assay is performed in plasmid man-
ufacturing on a routine base as in-process control, to
identify and monitor sensitive process steps, where shear
forces may damage plasmid DNA (e.g. sterile filtration).
Recently, this CGE technology has also been applied in
process development to monitor the removal of undesired
topologic plasmid variants from the supercoiled fraction
(for demonstration, see Figure 2).
Quality assurance and quality control
of plasmid DNA vectors
Plasmid DNA quality mainly depends on the type of man-
ufacturing, storage and application. The safety of such
drugs is dependent on vector construction, characteri-
zation and testing by toxicology and functional studies
before clinical trials. Driven by the production process,
those parameters are well defined, but subject to ongoing
improvements regarding the state-of-the-art on analytical
J Gene Med 2004; 6: S45–S53.
S48 M. Schleef and T. Schmidt

( a)

ccc monomer linear monomer oc monomer

(A) (C) (E)

ccc dimer linear dimer oc dimer

(B) (D) (F)

( b)
30

A
Fl uorescence / R FU

20
C

E
10

B
F
D

0
20 25 30 35
Mi grati on ti me, t m i g / mi n

Figure 1. CGE analysis of plasmid DNA. The analysis allows the identification and quantification of individual topology variants
of plasmid vector DNA and is the base of in-process-control and batch release control in plasmid manufacturing. (a) Selection of
different possible plasmid structures and (b) electropherogram (EPG) of a plasmid DNA sample with different topology variants

(a) M 1 2 3 4 M
(b) 25

ccc monomer
20
Fluorescence / RFU

15
oc form ccc dimer
10
ccc form
oc forms
5

0
0 10 20 30 40 50
Migration time, tmig / min

Figure 2. Production of ccc-supercoiled DNA in ccc-Pilot Grade quality. (a) AGE of a standard DNA sample (lanes 1, 2) and
ccc-Pilot-Grade DNA sample (lanes 3, 4). (b) CGE analysis of the ccc-Pilot-Grade DNA sample identifies more than 98% of ccc DNA

Copyright  2004 John Wiley & Sons, Ltd. J Gene Med 2004; 6: S45–S53.
Production of ccc-Supercoiled Plasmids S49

Table 1. Important criteria for quality assurance and quality In plasmid production, cellular RNA from E. coli is a major
control of plasmid DNA medicines (selection) contaminant, although RNA has a short half-life, because
Test Analytical method RNA blocks the binding capacity in all chromatography
steps. Therefore, an enzymatic digestion of RNA prior to
1. DNA concentration UV-absorption (260 nm) the chromatographic step is usually applied [e.g. 33–35].
2. General purity UV-scan (220–320 nm) In pharmaceutical manufacturing processes, the use of
3. Homogeneity (ccc content) CGE (capillary gel electrophoresis)
4. Purity (visible) Visual inspection bovine RNase is critical. In general, avoiding the use of
5. Purity (genomic DNA) Agarose gel (visual); Southern blot; RNAse increases the quality of the product. If bovine
quantitative PCR RNase A is used the origin of the pancreas has to be
6. Purity (RNA) Agarose gel (visual); fluorescence
assay; quantitative PCR assessed and certified. In literature, only a few plasmid
7. Purity (protein) BCA test purification processes completely avoid the use of RNase.
8. Purity (LPS) LAL test In these cases, the RNA is removed by specific precipi-
9. Purity (microorganisms) Bioburden test; sterility test
10. Identity (vector structure) Restriction fragment length conforms tation techniques [36–38]. However, they are applicable
to reference in AGE (1–3 enzymes) on a laboratory scale only.
11. Identity (sequence) Sequencing (double strand) Recent developments avoid the use of RNase through-
out the whole process – even in large-scale production
processes for GMP-Grade DNA [39]. With respect to prod-
techniques. Table 1 shows a selection of relevant qual- uct safety, no other enzymes are used and the cultivation
ity control tests for in-process-control (IPC) and product process is carried out without any animal-derived sub-
release. No guideline exists, indicating a certain value stances.
or specification for clinical material (except of ‘sterile’ for
sterility testing and ‘identical’ in case of sequencing DNA).
Regulatory bodies usually request a safe, state-of-the-art Genomic DNA
manufactured and well-controlled product.
A major impurity in plasmid DNA preparations deriving
from the plasmid-producing cells is commonly the host
Animal-free DNA production chromosomal or genomic DNA (gDNA). It has to be
removed completely by the process steps applied. While in
Process elements and cultivation media are a potential the past contaminations of plasmid DNA with gDNA were
source for contamination. Culture media for the growth in the range of 5–10%, novel purification technologies
of microorganisms were developed in the 19th and 20th allow a reduction to <1% gDNA. The most critical step
centuries. Liebig extracted beef and concentrated the is the alkaline lysis, where the gDNA as well as the
essence for storage and later use in media preparation plasmid DNA are denatured by a pH shift to alkaline
for the cultivation of microorganisms. The history of the buffer conditions. A subsequent neutralization step allows
development, manufacture and control of microbiological the reannealing of plasmid DNA within a short time.
culture media was summarized by Bridson [32]. The gDNA, however, does not reanneal completely to a
One major improvement for such media was the DNA double strand again. Therefore, the major part of
addition of peptones and salts, which led to the increased gDNA will be a component of the flaky material which
supplementation with amino acids and an enhanced is generated by the alkaline lysis. It mainly consists of
osmolarity. These peptones were generated by enzymatic potassium dodecyl sulfate, insoluble proteins, cell debris,
digest of meat. lipopolysaccharides (LPS) and DNA. Chromosomal DNA
Today’s technology for the generation of complex is extremely shear-sensitive, which can easily result in
bacterial growth media uses soy bean peptones to avoid DNA fragmentation. Some gDNA fragments are large
animal-derived protein sources. If it is indicated to enough to migrate in one distinct band in agarose
avoid any potential source of animal-derived components gel electrophoresis (AGE). Smaller fragments can be
within a plasmid manufacturing process, synthetic growth detected as an undefined smear by overloading the
media should be favored. However, only little data are agarose gel. More sensitive assays like Southern blot
available on cultivation processes with the intention of hybridization [40] can demonstrate this – depending on
manufacturing plasmid DNA by using synthetic growth the hybridization and washing conditions applied. The
media and being able to ensure at least comparable yields most sensitive assay is a kinetic PCR method using a
of product. If complex components are to be used, it TaqMan probe to quantify gDNA contaminations [41].
should be considered that no enzymes of animal origin
are applied (e.g. pepsin, pancreatin or trypsin). Hydrolysis
with acids avoids the use of such enzymes but leads to Other host cell impurities
higher salt content.
Typical and critical process elements in DNA purifica- Proteins, RNA as well as lipopolysaccharides (LPS,
tion are ribonucleases, like RNase A, which is typically endotoxins) are other host cell impurities which have
prepared from bovine pancreas. RNase A is able to to be removed to a minimum concentration during the
hydrolyze phosphodiester bonds within RNA molecules. purification process of plasmid DNA. The presence of

Copyright  2004 John Wiley & Sons, Ltd. J Gene Med 2004; 6: S45–S53.
S50
proteins can be detected by colorimetric assays, like the
Bradford test or the BCA test (bicinchoninic acid).
Quantification of residual RNA is important since
plasmid DNA is purified without using RNase. It can
be performed directly by fluorescence assays (Ribogreen)
after digestion of the plasmid DNA with DNase or after
AGE. A novel technique is the determination of RNA by
quantitative RT-PCR.
Bacterial LPS endotoxins have a pyrogenic effect on
mammalian cells. A dramatic reduction in this impurity
is necessary in order to use the manufactured DNA in
Research & Clinics. LPS can be determined by kinetic
measurement of Limulus polyphenus lysate (LAL) reaction
with endotoxins.
Plasmid identity
Plasmid DNA should be tested for identity. A simple
analytical method for determining plasmid identity is
restriction digestion of the plasmid DNA followed by AGE.
The length of the restriction fragments can be estimated
by comparison with a linear DNA size marker (e.g. 1-kb
ladder). The determined fragments have to conform to the
calculated fragments or to a reference DNA with respect
to identity. In our experience, four different enzymes each
with a minimum two restriction sites should be used.
The nucleotide sequence has to be determined by
sequencing the plasmid DNA. If the complete plasmid
or the coding region only has to be sequenced, this has to
be evaluated for each case.
Plasmid topology (structural
homogeneity)
Plasmids of identical nucleotide sequence isolated from E.
coli may exist in different shapes and forms. The structural
homogeneity of plasmid DNA is usually determined by
AGE. Different bands in AGE of a plasmid sample may
be assigned to different plasmid forms. The assignment
of bands to the different topologies, however, is not
easy since the electrophoretic mobility of plasmids of
different shape changes with the electrophoretic operating
conditions [42–45]. In addition, the quantification of
forms based on the signal intensity of stained bands in AGE
may not be reliable due to no linear responses. Adequate
equipment is required in order to obtain reproducible
results.
It is well known that typically only one band, the ccc
form, is observed when only a small amount of a plasmid
sample is applied to an agarose gel. Standard AGE usually
reveals two prominent bands: the ccc form and another
slower migrating form, commonly thought to be the oc
form. It has been demonstrated [30] that this is not always
the case, since the oc form co-migrates with ccc dimers.
Capillary gel electrophoresis (CGE) allows the iden-
tification and quantification of all prominent forms
mentioned [30,46]; see also [47]. CGE is performed
Copyright  2004 John Wiley & Sons, Ltd.
M. Schleef and T. Schmidt
using thin (100 µm) coated capillaries with a length
of 40–60 cm filled with a liquid polymer, e.g. solution
of hydroxypropylmethylcellulose. Electrophoretic separa-
tion takes place by applying a high voltage (5–30 kV)
between both ends of the capillary dipped in buffer reser-
voirs. Special intercalating dyes, like YOYO, YO-PRO or
TOTO, enable on-line detection with high resolution of
the different plasmid forms by laser-induced fluorescence
(LIF). The automated system offers high reproducibility,
reliable quantification and short analysis time. In contrast
to AGE, quantification of plasmid forms by using CGE is
possible over a wide range of linearity and needs only a
small amount of plasmid DNA (50 ng).
Plasmid stability
The physical and chemical stability of plasmid DNA is
a requirement to develop DNA-based pharmaceuticals,
which can be stored, shipped and applied even
under critical environmental conditions. The DNA
delivery sometimes requires the protection of this active
pharmaceutical ingredient, which is an issue of DNA
formulation. Guidance on the storage of plasmid DNA
can be found in the ICH guideline ‘Stability testing of new
drug substances and products’ – Q1A (R2) of 06. February
2003 [48].
Stability in plasmid vector applications
The use of gene-gun or jet-injection devices has been
developed in recent years for plasmid DNA drugs.
However, it requires further evaluation with respect to
plasmid stability. Monitoring the changes from ccc into
damaged oc or linear topologies during the application,
as well as optimizing the applied pressure with respect to
different tissues, plasmid size, buffers and intended depth
of injection, are essential [49].
The analysis of the topology of plasmids by CGE applied
for gene therapy or DNA vaccination [31] allows for
the first time the determination of the integrity and
distribution of the topology of the API. This issue is
of importance not only with respect to pharmaceutical
aspects of the homogeneity of the API, but also allows
optimization of the application process with respect to
the API integrity.
To evaluate potentially destructive forces on plasmid
DNA by jet-injection, CGE analysis of the pCMVβ plasmid
before and after jet-injection was performed [49].
For CGE of the original DNA before filling, samples
from the injector’s reservoir and from the ejected DNA
were analyzed. The plasmid pCMVβ was ejected from the
jet-injector at different pressures (2.0, 2.5 and 3.0 bar)
and compared with a reference. The plasmid topologies
resulting from the electropherograms are summarized in
Table 2. The results indicate that the use of such devices
will result in destruction of the ccc plasmid form by
ejection of the DNA solution. The damage increases with
J Gene Med 2004; 6: S45–S53.
Production of ccc-Supercoiled Plasmids S51

Table 2. Plasmid topology distribution of untreated pCMVβ Storage stability of plasmid DNA
sample and after jet-injection of pCMVβ at different pressures
[49]
Recent studies [50] show in detail potential alterations
Sample Sample description ccc-form oc-form of plasmid DNA by different storage conditions, such
Sample 2 DNA before filling into sample space 97.6% 2.4% as storage at −80 ◦ C or +4 ◦ C over a period of 0,
Sample 4 DNA from sample space after 93.5% 6.5% 1, 2, 3, 4, 5, 6, 7, 9 and 13 months (Figures 3
injection at 2.0 bar and 4). Figures 3a–3e show a representative series of
Sample 5 DNA from sample space after 85.4% 14.6%
injection at 2.5 bar electropherograms (EPG) generated by CGE analysis
Sample 6 DNA from sample space after 81.6% 18.4% of pCMVβ plasmid DNA. Storage of plasmid DNA at
injection at 3.0 bar −80 ◦ C shows no alteration concerning the plasmid
Sample 7 Ejected DNA after injection at 2.0 bar 93.3% 6.7%
Sample 8 Ejected DNA after injection at 2.5 bar 84.6% 15.4% forms from the intact ccc forms to the damaged oc
Sample 9 Ejected DNA after injection at 3.0 bar 77.2% 22.8% forms (Figures 3b and 3d). A very different situation
is obvious for pCMVβ plasmid DNA stored at +4 ◦ C
(Figures 3c and 3e). During storage the amount of
ccc monomer and the dimeric ccc form decreased
increasing pressure. In addition, we found that also the dramatically resulting in increasing amounts of the
non-ejected DNA had been destroyed from the ccc to the damaged oc and linear forms. This strongly indicates that
oc form, which happened also at higher pressure values such storage conditions promote plasmid DNA instability,
predominantly. We assume that turbulences within the which could have an impact on transfection efficiency.
device are responsible for this effect. Although still controversial, there is an understanding

(a) 30
ccc Monomer
Fluorescence / RFU

20

ccc Dimer
10

oc Forms
0

0 10 20 30 40 50
Migration time, tmig / min

(b) 60 ccc Monomer (c) 60 ccc Monomer

Fluorescence / RFU

Fluorescence / RFU

50 50

40 40

30 30
ccc Dimer ccc Dimer
20 20

10 oc Forms 10 oc Forms

0 0
0 10 20 30 40 50 0 10 20 30 40 50
Migration time, tmig / min Migration time, tmig / min

(d) 50 ccc Monomer (e) 40

Fluorescence / RFU

oc Forms
Fluorescence / RFU

40
30
30
20 ccc Monomer
20 ccc Dimer linear Forms

10 oc Forms 10

0
0
0 10 20 30 40 50 0 10 20 30 40 50
Migration time,tmig / min Migration time, tmig / min

Figure 3. Representative electropherograms (EPG) of storage CGE analysis of a pCMVβ plasmid. The EPG show the analysis of
pCMVβ plasmid DNA samples stored at −80 ◦ C or 4 ◦ C for 1, 2 and 13 months. (a) Control material at the beginning of storage;
(b) sample stored 1 month at −80 ◦ C; (c) sample stored 1 month at 4 ◦ C; (d) sample stored 13 months at −80 ◦ C; and (e) sample
stored 13 months at 4 ◦ C. From [50]

Copyright  2004 John Wiley & Sons, Ltd. J Gene Med 2004; 6: S45–S53.
S52
ccc monomer ccc dimer oc forms
(a) 100
80
Amount / %
60
40
20
0
0 2 4 6 8 10 12
Storage time / months
cccmonomer ccc dimer oc forms
(b) 100
80
Amount / %
60
40
20
0 levels. Firstly, the vectors have to be more efficient (on
0 2 4 6 8 10 12
Storage time / months
Figure 4. Quantitative analysis of plasmid DNA topologies by
CGE. Plasmid samples were stored at −80 ◦ C (a) and 4 ◦ C (b)
over a period of up to 13 months and analyzed by CGE. The
quantitative CGE analysis data are indicated as the percentage
of the respective ccc and oc forms. From [50]
by many investigators that intact supercoiled DNA is
advantageous for efficient gene transfer over open
circular or degraded linear DNA [51]. Therefore, a
specification of DNA used for gene therapeutic purposes
is essential to clearly define the gene therapeutic product
and is an important requirement if clinical studies are
planned. Additionally, in vivo data were generated by jet-
injection technology for intratumoral in vivo gene transfer
[16]. DNA of the pCMVβ plasmid stored for 1, 2 or
13 months at −80 ◦ C was used for jet-injection of nude
mice carrying xenotransplanted human colon carcinoma.
The animals received five jet-injections intratumorally,
representing a total 50 µg of naked plasmid DNA
per animal. It has been demonstrated [51] that the
storage of this plasmid could not influence gene transfer
efficiency, which was determined quantitatively by β-
galactosidase ELISA and qualitatively by histochemical
X-gal staining.
Thus, for non-viral gene therapy approaches stable
quality of the used plasmid DNA is an essential pre-
requisite for reproducible and efficient gene transfer.
Therefore, apart from the CGE analysis of plasmid DNA
stored for different time periods at −80 ◦ C, evaluation
of its suitability for gene transfer is required. We
recently analyzed plasmid DNA of the batch shown in
Figure 3a by CGE, that was stored at −20 and −80 ◦ C
for 30 months. Also these samples did not show any
Copyright  2004 John Wiley & Sons, Ltd.
M. Schleef and T. Schmidt
detectable ccc degradation. The storage of DNA at
other temperatures was tested by accelerated studies
(PlasmidFactory, unpublished).
A major improvement of clinical plasmid applications
is an ‘on-site CGE sampling’, where a sample of the DNA
used for any critical application is tested immediately
after use by CGE to determine its status at the moment
of use. This information is at least as relevant as the
quality data obtained at the moment of producing the
DNA pharmaceutical.
14
Future developments
linear form
Until now, plasmid DNA in pharmaceutical development
has been used to design the coding gene used for the
production of a therapeutic recombinant protein. Further
applications are the use of plasmid vectors for the
production of viral particles, where plasmids in some
applications may be part of the pharmaceutical. The
direct application of DNA for vaccination of compensation
(gene therapy) requires further development at different
14
the level of gene transfer and expression). Secondly,
the vectors have to be targeted to reach the cells of
interest and, at least, the production quality needs to
be extended to aspects dealing with the isomeric and
inefficient forms as well as potentially harmful process
components (e.g. RNase). Another aspect in the future
should be the application of vectors without antibiotic
resistance genes. The stable production of these vectors
will be a challenge.
The storage and delivery of DNA drugs are frequently
involved with drug formulation in defined (buffer) media.
Typically, a plasmid DNA solution will be frozen for long-
term storage as the final critical step where the formation
of ice crystals may damage the DNA. Optimization
and storage validation studies are possible now, since
CGE is available. Even lyophilization of DNA can be
produced and may allow a reduction in costs for storage
and logistics. PlasmidFactory started research projects to
further increase vector safety: after the complete exclusion
of any antibiotic and any animal-derived substances from
the whole plasmid manufacturing process, we removed
also defect plasmid vector topologies and bacterial
genomic DNA from this pharmaceutical. Next steps in
our development are the stabilization of plasmids (e.g.
large and fragile types) in storage and application and the
development of vector systems to improve expression and
gene transfer efficiency.
Acknowledgements
We thank Erwin Flaschel, Carsten Voß, Kirsten Lanfermann
and Thomas Schäffer, University of Bielefeld, Faculty for
Technology, for their support with the research projects on DNA
manufacturing, and the QC team of PlasmidFactory, Bielefeld,
Germany, for their contribution.
J Gene Med 2004; 6: S45–S53.
Production of ccc-Supercoiled Plasmids
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