Production of Plasmid DNA in Bioreactors 295

21
A Simple Method for the Production of Plasmid DNA
in Bioreactors

Kristin Listner, Laura Kizer Bentley, and Michel Chartrain

Summary
The need for large quantities of purified plasmid DNA has increased as the applications of
DNA vaccines continue to expand. This chapter describes a simple, scaleable procedure based
on the fed-batch cultivation of various Escherichia coli clones, which can be easily imple-
mented and scaled-up to large bioreactors. Although some clones may require minor modifica-
tions to the feeding strategy, in general, this procedure, implemented as described, is likely to
support the production of milligram to gram quantities of plasmid DNA.
Key Words: Plasmid DNA; E. coli; fermentation; fed-batch: DNA vaccines.

1. Introduction
The use of plasmid DNA vaccines is rapidly gaining popularity in various
treatment regimens including prophylaxis against both microbial and viral
infections, gene therapy applications, and the control of cancer cell prolifera-
tion (1–11). Although the technology has encountered set backs linked to low
potency, recent advances in both formulation and novel delivery methods have
given DNA vaccination additional opportunities (12–14). At this time, it
remains a promising therapy as an equine West Nile vaccine is likely to be the
first DNA vaccine to gain Food and Drug Administration (FDA) approval in
the very near future (15). Because the amount of plasmid DNA required for the
injection of each patient is large by vaccine standards, on the order of several
milligrams per dose, technological and economical pressures are correlatively
placed on production methods (16). Consequently, the production of sufficient
amounts of material for laboratory, preclinical, and clinical studies can rapidly
become a bottleneck unless efficient and economical manufacturing procedures
are implemented.

From: Methods in Molecular Medicine, Vol. 127: DNA Vaccines: Methods and Protocols: Second Edition
Edited by: W. M. Saltzman, H. Shen, J. L. Brandsma © Humana Press Inc., Totowa, NJ

295
296 Listner et al.

Routinely, plasmid DNA is produced in the bacterial host, Escherichia coli.

A survey of the literature indicates that plasmid production can be supported
by many of the strains typically used for the expression of recombinant pro-
teins (17). Additionally, cultivation of the host bacterium can be performed in
many medium formulations, ranging from chemically defined to complex and
undefined, with media of both types supporting plasmid production (17).
The requirement for small amounts of purified plasmid DNA for laboratory
studies can be satisfied by the cultivation of E. coli in large shake flasks, a
fairly simple process. However, the linear scale up of this type of process is
rather inefficient and cumbersome because it rapidly becomes labor and time
intensive. Alternatively, the use of well-controlled bioreactors can alleviate
many of the limitations of shake flask cultivation. In particular, the control of
dissolved oxygen and pH in bioreactors, together with the implementation of
fed-batch strategies, allows for significant biomass increases when compared
to shake flask cultures (18–20). The increase in biomass achieved in the
bioreactors often correlates with higher plasmid yields. Nevertheless, one
should keep in mind that final volumetric (g/L) and specific (mg/g of biomass)
plasmid yields intrinsically depend on many variables including the vector con-
struct, host cell line, medium composition, and cultivation conditions. This
chapter will address the latter two points, whereas the former topics are
reviewed in other chapters.
Described in this chapter is a simple, scaleable method that can be easily
implemented in support of the production of hundreds of milligrams to several
grams of plasmid DNA. This procedure requires the use of bioreactors that can
provide appropriate oxygen transfer and good control of environmental condi-
tions such as pH and temperature. Any typical well-instrumented laboratory
scale bioreactor is suitable for the implementation of this procedure. Further-
more, these methods can be scaled up to large-scale bioreactors without major
modifications.

2. Materials
1. E. coli DH12s host strain (Invitrogen, Carlsbad, CA).
2. Basal cultivation medium: (NH4)2SO4, 3.0 g/L, K2HPO4, 3.5 g/L, KH2PO4, 3.5 g/
L, yeast extract (BD/Difco, NJ), 10.0 g/L, HySoy Peptone (Sheffield Products,
NY), 10.0 g/L, UCON LB625 Antifoam (Dow Chemical, MI), and 1.0 mL/L in
distilled water, pH 7.1.
3. 500 g/L Glucose solution. Autoclave sterilize at 121qC for 30 min.
4. Medium supplement solution: thiamine-HCl, 24.0 g/L, MgSO4x7H2O, 240.0 g/
L, neomycin sulfate, 9.6 g/L in distilled water. Filter-sterilize through a 0.2-Pm
membrane.
5. Trace elements solution: FeCl3x6H2O, 27.0 g/L; ZnCl2, 2.0 g/L; CoCl2x6H2O,
2.0 g/L; Na2MoO4x2H2O, 2.0 g/L; CaCl2x2H2O, 1.0 g/L; CuCl2x2H2O, 1.3 g/L;
Production of Plasmid DNA in Bioreactors 297

and H3BO3, 0.5 g/L, 1.2N HCl, 100 mL/L, prepared in distilled water. Filter-
sterilize through a 0.2-Pm membrane.
6. 600 g/L Glucose solution. Autoclave sterilize at 121qC for 30 min.
7. 370 g/L Yeast extract solution. Autoclave sterilize at 121qC for 30 min.
8. 40% Glycerol solution (w/v). Autoclave sterilize at 121qC for 30 min.
9. 15% Phosphoric acid (v/v).
10. 30% Sodium hydroxide (v/v).
11. STET Buffer: 8.0% sucrose; 50 mM Tris-HCl, 100 mM ethylene-diamine
tetraacetic acid (EDTA), 2.0% Triton X-100, pH 8.5. Filter-sterilize through a
0.2-Pm membrane.
12. 4 g/L Lysozyme solution: 4.0 mg lysozyme/mL STET buffer.
13. RNace-It Cocktail (Stratagene, CA).
14. Gen-Pak FAX Anion Exchange Column, 4.6 u 100 mm (Waters Corporation,
MA).
15. High-performance liquid chromatography (HPLC) Buffer A: 25 mM Tris-HCl, 1
mM EDTA, pH 8.0.
16. HPLC Buffer B: 1 M NaCl, 25 mM Tris-HCl, 1 mM EDTA, pH 8.0.
17. HPLC Buffer C: 0.04 M H3PO4.
18. 0.45 Pm u 47 mm Nitrocellulose filters (Millipore, Bedford, MA).
19. 250-mL Baffled shake flasks.
20. 2.8-L Baffled Fernbach shake flasks.
21. 15.0-mL Falcon sterile tubes.
22. 500-mL Nalgene sterile media bottles.
23. Thermomixer (Eppendorf Westbury, NY).
24. Filtration manifold assembly (Gelman, cat. no. 4205).
25. 2.5-mL Microcentrifuge tubes.

3. Methods
The methods described herein outline the process for large-scale production
of plasmid DNA using the E. coli DH12s strain. This process has proven to be
extremely efficient in the production of hundreds of milligrams to gram quan-
tities of several plasmid constructs. Various plasmids, all based on the V1Jns
backbone (21,22) and ranging in size from 7 to 9 kb pairs, have been success-
fully produced by this method (see Note 1).
The following sections will describe (1) the preparation of the cultivation
medium and feed solutions, (2) construction of the E. coli cell banks, (3)
execution of the production process, and (4) sample preparation and analytical
methods.
3.1. Medium Preparation
The cultivation medium used in this process is a complex medium that is
used for both shake flask and bioreactor cultivation of E. coli (Table 1). For
shake-flask cultures, the basal batch medium and medium supplements may be
298 Listner et al.

Table 1
Complex Cultivation Medium for Shake
Flask Seed Stages
Medium component Concentration

(NH4)2SO4 3.0 g/L

K2HPO4 3.5 g/L
KH2PO4 3.5 g/L
Yeast extract 10.0 g/L
HySoy peptone 10.0 g/L
antifoam (UCON LB625) 1.0 mL/L
Glucose 5.0 g/L
Thiamine-HCl 0.20 g/L
MgSO4x7H2O 2.0 g/L
Neomycin sulfate 0.08 g/L
Trace elements 1.0 mL/L

prepared in advance and stored appropriately for use as needed. (The basal
medium and trace elements solutions may be kept at 4qC, although it is recom-
mended that the supplement solution be frozen between –20 and –70qC.) For
bioreactor cultivation, the basal medium is prepared in the fermentor and in
situ sterilized immediately before use. The medium supplements are prepared
separately, filter-sterilized, and then added to the bioreactor poststerilization.
The feed solution for the fed-batch process requires careful preparation, as the
concentrations of its components are near solubility limits.
3.1.1. Shake Flask Medium Preparation
1. Prepare the basal cultivation medium (see Subheading 2., item 2) by dissolving
the first three ingredients in approximately two-thirds the total volume of dis-
tilled water.
2. Add the yeast extract and soy peptone components. Mix the solution with low
heat until completely dissolved. Ensure that the pH of the solution is neutral, and
tare the volume with distilled water.
3. Autoclave sterilize the solution at 121qC for 25 min. (For shake flask cultures,
the basal medium can be prepared in advance and stored at 4qC for a few days.)
4. Dissolve all components of the medium supplement solution (see Subheading
2., item 4) in distilled water. Filter-sterilize through a 0.2-Pm membrane. (Be-
cause of stability concerns regarding thiamine-HCl and neomycin sulfate in solu-
tion, the medium supplement solution is either prepared fresh, immediately before
use, or it can be prepared in advance and stored in small aliquots at –70qC.)
5. Prepare fresh or thaw a vial of prepared medium supplement solution, and asep-
tically add 8.3 mL/1 L of basal cultivation medium.
Production of Plasmid DNA in Bioreactors 299

6. Aseptically add 10 mL/L of the sterile 500 g/L glucose solution (see Subheading
2., item 3).
7. Add 1.0 mL/L of the trace elements solution (see Subheading 2., item 6). Sterile
antifoam may be added at a concentration of 1.0 mL/L if necessary.

3.1.2. Bioreactor Medium Preparation

The description for preparation of a 30-L bioreactor, with a working volume
of 15 L basal cultivation medium, is as follows:
1. The basal cultivation medium (including the antifoam) is prepared and sterilized
in the bioreactor at 123qC for 25 min.
2. Once the reactor has cooled to 37qC, aseptically add the presterilized medium
supplements to the bioreactor: 10.0 mL/L of 500 g/L glucose solution, 8.3 mL/L
medium supplement solution, and 1.0 mL/L trace elements solution. In order to
facilitate transfer into the bioreactor, all three supplement solutions can be com-
bined at this stage.
3. Set up the acid and base control solutions on the tank. Adjust the pH of the pre-
pared medium to 7.1 prior to inoculation.

3.1.3. Feed Solution Preparation

The nutrient feed solution consists of a mixture of 40% glucose and 10%
yeast extract. It is prepared from two concentrated stock solutions. The solu-
tions are prepared separately in order to prevent “browning” of the mixture
during sterilization.
1. Prepare the 600-g/L glucose solution by slow addition of the glucose in distilled
water and mixing with low heat.
2. Prepare the 370-g/L yeast extract solution by slow addition of the powder in
distilled water and mixing with low heat.
3. Autoclave the solutions at 121qC for 30 min.
4. When cooled, mix the glucose and yeast extract solutions at a ratio of 2.5:1 (v/v)
to yield the complete feed solution.

3.2. Cell Bank Preparations

Expansion of the E. coli plasmid-containing clone of interest from a master
cell bank is strongly recommended, as it allows for consistent process perfor-
mance. Two frozen cell banks are prepared—the Master Seed and Working
Seed banks. The Master Seed is stored in cryovials or tubes and should be
regarded as the original cell source. The Working Seed bank is stored in bottles,
which are used to directly inoculate the production bioreactor upon thaw. This
one-stage process eliminates the need for a shake flask inoculum stage in order
300 Listner et al.

Fig. 1. Schematic for the preparation of master and working cell banks. The Master
Seed (MS) bank is prepared by inoculating a shake flask with the source clone. The
cells harvested during mid-exponential growth (as determined by OD) are mixed with
glycerol to give a 20% final glycerol concentration in the cell suspension. The resulting
glycerol stock culture is then dispensed into cryotubes (10-mL aliquots) and frozen at –
70qC. The Working Seed (WS) bank is prepared by inoculating a shake flask with a
frozen cell suspension from the MS bank. The cells harvested in mid-exponential
growth are mixed with glycerol to give a 20% final concentration. The culture mixture
is then dispensed into sterile Nalgene bottles (300-mL aliquots) and frozen at –70qC.

to greatly simplify and streamline operations. Figure 1 provides a pictorial

schematic of the procedure.
3.2.1. Master Seed Bank
1. Prepare a sterile, 250-mL baffled shake flask containing 30 mL of the complex
medium previously described.
2. Inoculate the broth with approx 10 PL of cells from a glycerol stock of the source
clone or from a single colony isolated from a selective agar plate.
3. Cultivate at 37qC, with shaking at 220 rpm, until an optical density OD600 value
of between 4.0 and 9.0 is achieved. This range is typically representative of the
mid-exponential growth phase, although proper growth kinetic analysis of the
culture should be performed to determine if this range is suitable for the clone of
interest.
4. Prepare the cell bank by mixing an equal volume of culture with cold, sterile 40%
glycerol.
5. Dispense the mixture into 10-mL vials to yield the master cell bank (MB) and
store the vials at –70qC.
Production of Plasmid DNA in Bioreactors 301

3.2.2. Working Seed Bank

1. Thaw one MB vial at room temperature.
2. Prepare a sterile, 2.8-L baffled Fernbach shake flask with 500 mL of the com-
plete complex medium as previously described.
3. Inoculate the flask with 10 mL of seed culture from the thawed MB vial.
4. Cultivate at 37qC, with shaking at 180 rpm, until an OD600 value ranges from 4.0
to 9.0 is achieved (mid-exponential growth phase).
5. Add an equal volume of culture to cold, sterile 40% glycerol and mix rapidly.
6. Dispense 300 mL aliquots into sterile 500-mL Nalgene bottles. The resulting WB
is stored frozen at –70qC.

3.3. Fermentation Process

The fed-batch fermentation process outlined next incorporates the use of
standard laboratory-scale bioreactors. Fermentors, which can provide accurate
control of environmental conditions such as pH and temperature, as well as good
oxygen transfer for efficient cell culture growth, are key for ideal process perfor-
mance. Figure 2 provides a schematic of the production bioreactor set-up.
The plasmid production process consists of two distinct phases. The first
phase of the process is characterized by rapid cell expansion through the utili-
zation of the batched nutrients in the basal medium. Typical exponential growth
kinetics is observed during this phase of the process (see Fig. 3A). This phase
is characterized by an elevated demand for oxygen and, in the absence of a
computer-controlled system, will require the operator to closely monitor the
bioreactor. This is to ensure that the set-points for mixing, air flow, and back
pressure are adequately increased in order to maintain the dissolved oxygen
(DO) at the desired level (30–40% of atmospheric saturation).
The feeding is initiated during the mid-exponential growth phase. Once all
of the excess nutrients are consumed, the oxygen demand is directly dependent
on the needs for consumption of the nutrients delivered to the cells via the
feeding solution. Because the nutrient delivery rate is kept at a relatively low
rate, the oxygen uptake rate (OUR) is maintained at a low and relatively con-
stant value during the entire duration of the feeding phase (see Fig. 3B). This
second phase is characterized by a lower growth rate, which is conducive to
plasmid amplification (23–26). The process takes about 1 d to complete. Dif-
ferences in the growth rates of various clones are to be expected and may result
in either longer or shorter process durations.
Specifically, the production bioreactor, containing 15 L of cultivation
medium prepared as described above, is inoculated with 300 mL of a thawed
working seed bottle. This volume translates into a 2.0% (v/v) inoculum, a rela-
tively high ratio that allows for a short lag phase and thus an overall rapid
process. The initial cultivation conditions are as follows: temperature, 37qC;
302 Listner et al.

Fig. 2. Overview of the bioreactor set-up for an Escherichia coli fed-batch plasmid
production process. The bioreactor is inoculated directly with one bottle (300 mL) of
thawed cell suspension from the Working Cell bank (representative of a 2% inoculum
in a 15-L working volume). Dissolved oxygen and pH are monitored online. pH is
maintained at 7.1 through automatic addition of H3PO4 and/or NaOH solutions. DO is
maintained at 30–40% of initial saturation via the automated control of the agitation.
Once mid-exponential growth is reached (measured on-line via Carbon Evolution Rate
or off-line by Optical Density determination), the feeding of a nutrient solution, made
up of yeast extract and glucose, is initiated.

back pressure, 4.5 psig; airflow, 7.5 slpm (0.5 vvm). Carbon dioxide evolution
rate (CER) and OUR are measured on-line via the use of a mass spectrometer
(Prima V Mass Spectrometer Model 600, Thermo ONIX Corp., Houston, TX).
The DO is maintained to a set point greater than or equal to 30–40% of air
saturation by automatic cascade control of the agitation speed (between 250
and 750 rpm). The pH is maintained at 7.1 r 0.1 by feedback-controlled auto-
matic addition of 15% phosphoric acid or 30% sodium hydroxide solutions
throughout the fermentation cycle.
After approx 6 h of cultivation, mid-exponential growth is reached, as indi-
cated by a CER of approx 20 mmol/L/h (or by an optical density at 600 nm of
8–12). Feeding of the nutrient feed solution (40% glucose;10% yeast extract;
Production of Plasmid DNA in Bioreactors 303

Fig. 3. Typical cell metabolic and plasmid production profiles. (A) Baseline plas-
mid production in batch culture. Cells from a frozen Working Seed bank were inocu-
lated into a 30-L bioreactor containing 15 L of the complex cultivation medium (Table
1), and were allowed to grow batch-wise. Cultivation conditions were: pH 7.0, tem-
perature maintained at 37qC, and dissolved oxygen controlled to 30–40% of initial
saturation. OUR and CER peaked at approx 35 mmol/L/h, 6–8 h post-inoculation. The
decrease observed in both CER and OUR values at 8 h post-inoculation is a result of
the rapid decline in cellular metabolism as the batched nutrients were consumed by the
culture. Correlatively, a rapid increase in dissolved oxygen is observed. A maximum
specific plasmid yield of approx 4.5 Pg plasmid/mg DCW was achieved after 8 h of
304 Listner et al.

see Subheading 3.1.3.) is then initiated at a rate of 0.3 g solution/min/reactor

(1.2 g/L/h). To ensure that the oxygen demand of the cells is fully met through-
out the feeding phase, the airflow may be increased from 7.5 to 12.0 slpm, and
the back pressure from 4.5 to 15.0 psig, upon initiation of the feed. The agita-
tion remains under the control of the computer system to maintain the DO at
30–40% of atmospheric saturation. Plasmid amplification takes place during
the feeding phase of the process (see Fig. 3 and Note 2).
After approx 18 h of feeding, plasmid production reaches a plateau, and the
process is terminated (see Note 3). The content of the fermentor is then chilled
and held at 10qC until harvest takes place. It is possible to hold the chilled
broth for up the 24 h without observing any significant decreases in plasmid
yields. Sample volumes of 25–50 mL are periodically removed throughout the
fermentation and are used to measure plasmid production, biomass, and any
desired nutrient or metabolite.
3.4. Sample and Analytical Methods
3.4.1. Dry Cell Weight
1. Weigh two nitrocellulose filters on an analytical balance and record their weights.
2. Place the filters on a filtration manifold assembly that is connected to a vacuum
source. Prewet the filters using distilled water.
3. Add 5 mL of culture to each filter and rinse with 5 mL of distilled water. (When
the OD600 reaches values greater than 20, dilution of the broth may be needed in
order to achieve efficient filtration.)
4. Once the liquid is completely removed from the filter surface, turn off the vacuum
and remove the filters. Dry them in a household microwave oven at 50% power
for 10 min (this length of time may need adjustment based on the power of the
microwave oven used).
5. Weigh the dried filters on the analytical balance, and subtract the filter tare weight

Fig. 3. (continued) batch cultivation. (B) Fed-batch based fermentation process. Cells
from a frozen Working Seed bank were inoculated into a 30-L bioreactor containing 15
L of the complex cultivation medium (Table 1) and were allowed to grow batch-wise.
Cultivation conditions were: pH 7.0, temperature maintained at 37qC, and dissolved
oxygen controlled to 30–40% of initial saturation. Feeding of a nutrient solution (glu-
cose/yeast extract) at a rate of 1.2 g/L/h was initiated at mid-exponential growth (indi-
cated by a CER value of 20 mmol/L/h, approx 8 h post-inoculation). Nutrient feeding
allowed for a higher metabolic activity as indicated by an OUR peak at 90 mmol/L/h.
Following OUR peak, the constant feeding supported and sustained the cells at a slow
growth rate, as indicated by the maintenance of the OUR at approx 18 mmol/L/h for the
remainder of the cultivation cycle. Plasmid production ceased after about 12 h of feed-
ing. A final plasmid volumetric yield of 110 mg/L and a specific plasmid yield of 11.3
Pg/mg of dry cell weight were achieved with the fed-batch process.
Production of Plasmid DNA in Bioreactors 305

from the gross weight to get DCW (g/L) using the following calculation:

⎛ Average DCW per 5 mL (g) ⎞ ( ⎛ 1000 mL ⎞

Final DCW (g/L) = ⎜
⎝ ⎟⎠ dilution factor ) ⎜⎝ ⎟⎠
5 mL L

3.4.2. Sample Preparation and Cell Lysis

The optical density of samples taken from the fermentation production tanks
should be measured at 600 nm using a spectrophotometer. The volume of cul-
ture needed to prepare OD10 pellets is calculated by using the following equa-
tion:

10
Volume to add to tube (mL) =
measured OD 6000

1. Centrifuge the calculated volume of culture for 5 min at approx 15,000g in an

Eppendorf 5414 C centrifuge, or similar, and discard the supernatant.
2. Resuspend the OD10 pellet in 0.5 mL of STET buffer.
3. Add 0.5 mL of lysozyme solution and mix well.
4. Incubate the tubes for 45 min at 37°C in an Eppendorf Thermomixer R, or simi-
lar, with continuous shaking at 500 rpm.
5. Immerse in boiling water for 1 min.
6. Centrifuge the tubes for 15 min at 15,000g.
7. Pour the supernatant into HPLC vials and add 10 μL of RNase solution. Cap the
vials, shake to mix, and incubate for 1 h at room temperature.
8. Analyze the supernatant by anion-exchange chromatography to quantify the
amount of plasmid DNA present in each sample.

3.4.3. Anion Exchange HPLC

The separation of plasmid DNA is achieved using an HPLC system equipped
with a Gen-Pak FAX anion exchange column. Separation is obtained by using
a salt gradient consisting of Buffer A and Buffer B delivered at a rate of 0.75
mL/min. Buffer C is used to wash the column between injections. The gradient
is 70/30 (v/v) A:B for 2 min, followed by 35/65 (v/v) A:B for 13.5 min. The
column is then washed for 4.5 min with Buffer C, followed by a return to 70/30
(v/v) A:B for re-equilibration before performing the next injection. Detection
is performed at 260 nm, and plasmid DNA elutes after approx 5 min.
Specific plasmid DNA yield is reported as micrograms plasmid per milli-
gram dry cell weight, and volumetric plasmid yield as grams plasmid per liter.
Plasmid DNA standards of known concentrations, ranging from 50 to 200 μg/
mL, should be used to calibrate the HPLC for a linear range of 5 to 20 μg
plasmid (based on a 10-μL injection volume). In order to ensure that the lysis
306 Listner et al.

multiple experiments. It can serve as a tool to assist in the identification of

issues specific to either the lysis or the HPLC procedures.
3.5. Conclusion
The methods and examples presented in this chapter provide a simple,
scaleable procedure that can be easily implemented using any well-
instrumented laboratory scale bioreactor. The process described here will eas-
ily scale up to large bioreactors with minimal adjustments. These procedures,
with some minor optimization to the feed rate if needed, are likely to generate
sufficient plasmid yield when employing various E. coli clones. When culti-
vating a single clone, the implementation of this process from cell banking to
fermentation will ensure reproducibility between batches.

4. Notes
1. Construct diversity: an evaluation of process performance for different plasmid
constructs is presented in Table 3. The performance of three different clones,
harboring a V1Jns vector backbone with different gene inserts, shows consistent
results between constructs and between fermentation batches. Although a feed-
ing regimen of 1.2 g/L/h performed well for the three constructs shown here, it
may be prudent to evaluate the performance for each specific clone, especially if
growth is poor. Both the feed rate and duration of the feed stage are variables to
explore when working to optimize the process for a given clone. Nonetheless, the
implementation of this simple feeding strategy should translate well to most E.
coli isolates and will ultimately supply investigators with hundreds of milligrams
to gram quantities of the plasmid of interest.
2. Plasmid yield: details of a representative fed-batch fermentation process profile
are depicted in Fig. 3. The feeding stage is a critical phase of the process that
affects final product yields. Without feeding, the metabolic activity of the cells
abruptly ceases and is reflected by a rapid decrease in OUR, which occurs as
soon as the batched nutrients are completely consumed (see Fig. 3A). Correla-
tively, relatively low volumetric and specific plasmid yields are achieved. How-
ever, the implementation of a nutrient-feeding regimen for approx 18 henables
the cells to maintain metabolic activity, albeit at a lower level that that achieved
during exponential growth. The lower rate of growth achieved during this phase
of the cultivation allows for plasmid amplification as presented in Fig. 3B. In this
specific example, threefold increases in both specific and volumetric plasmid
yields are achieved. It must be noted that the improvement in specific yield also
positively impacts downstream processing. This is because lower plasmid losses
are observed during the purification procedure when working with a source cul-
ture with a high specific yield.
3. Nutrient feeding: in the same example, continuing the feeding beyond 24 h did
not support any additional benefits (see Fig. 3B). However, certain clones may
require an extension of the feeding phase to enhance production. It has also been
determined that the feed rate is a crucial factor in the amount of plasmid produc-
Production of Plasmid DNA in Bioreactors 307

Table 2
Sample Fermentation Results for One Plasmid Construct
Feeding Dry cell weight Volumetric yield Specific yield
condition (g/L) (g plasmid/L) (Pg plasmid/mg DCW)

No feeding 6.8 (T = 8 h) 0.03 4.7

1.2 g/L/h 9.9 (T = 24 h) 0.11 11.3
2.4 g/L/h 11.3 (T = 24 h) 0.07 6.6

Table 3
Sample Fermentation Results for Three Different Gene Inserts
Constructa
and plasmid Dry cell weight Volumetric yield Specific yield
size (g/L) at T=24h (g plasmid/L) (Pg plasmid/mg DCW)

1 (8.7 kb) 7.9 + 0.6 0.07 + 0.01 9.1 + 0.3

2 (8.7 kb) 8.6 + 0.1 0.10 + 0.01 11.6 + 1.4
3 (7.1 kb) 8.8 + 0.0 0.07 + 0.01 7.8 + 1.3
aNote: Constructs no. 1–3 represent three different genes cloned into the same

multiple cloning site of the V1Jns vector backbone. N = 2 fermentation batches per
gene construct in this summary.

tion from a particular clone. Table 2 displays comparative data from one clone
with different feeding strategies. A feeding regimen of 1.2 g/L/h rate was found
most appropriate in this case.

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