High-Cell-Density Culture to Produce Plasmid DNA for Gene Therapy in E. coli

ABSTRACT

The recombinant host Escherichia coli DH10B bearing the plasmid pIDKE2 was grown under fed batch conditions, and the effects of different medium components on plasmid yield and cell mass were evaluated at a 5-L fermentation scale. Results showed that glucose was the optimal carbon source at 265 g/L. After testing different levels of nitrogen, a defined complex medium was formulated for optimal plasmid production, capable of producing up to 0.44 mg plasmid DNA per g dry cell weight.

Interest in gene therapy has grown considerably over the last decade because of its promise as a treatment for the prevention, treatment, and cure of diseases such as cancer and acquired inmudeficiency syndrome (AIDS).^1,2 It has been shown that naked DNA injected into muscle tissue is expressed in vivo and that the introduction of immunogenic sequences can result in animal vaccination against the encoded peptide.^3,4 In general, DNA-based vaccines are considered very safe, in part because of the lack of genetic integration, and also because of the absence of a specific immune response to the plasmid itself.⁵ This safety makes the use of DNA vaccines very attractive.⁶ In addition, unlike live attenuated vaccines, plasmid DNA (pDNA) vaccines do not carry the hypothetical risk of reverting to a viable state and causing illness.⁷

(MSC. JORGE VALDÉS)

Gene therapy treatments require considerable amounts of pDNA, which must be homogeneous with respect to its structural form and DNA sequence. Certain growth conditions can lead to significant amounts of non-supercoiled pDNA forms. This means that the homogeneity and quality of the final pDNA product will be a function of interactions between the host, the plasmid, and the growth environment. Therefore, the choice of fermentation protocol will be critical to minimize process contaminants that need to be removed during downstream processing.⁸

A further feature of fermentation processes for large-scale plasmid production is the performance of high-density fermentations to obtain large amounts of biomass. Experimental work on the composition of bacterial growth media has demonstrated that fermentation conditions and growth media strongly influence the yield and quality of plasmids produced in E. coli cells.⁹

In this work, the medium composition of a high-cell-density culture for the production of plasmid pIDKE2 in a recombinant E. coli DH10B system was studied. The results provide a defined medium for pDNA production.

MATERIALS AND METHODS

Strain and Plasmid

The plasmid used in this work was pIDKE2 with a kanamycin-resistant marker; it was transfected into E. coli DH10B using standard methods.¹ The E. coli was then grown on several LB agar plates supplemented with 50 μg kanamycin/mL.

Culture Media

The basic LB medium for seed cultivation contained 10 g tryptone/L, 5 g yeast extract/L, and 10 g NaCl/L, supplemented with 50 μg kanamycin/mL. A fed-batch fermentation was carried out in a 5-L bioreactor with a working volume of 4 L, in a complex medium containing 5 g glucose/L, 1.4 g KH₂PO₄/L, 8.6 g (NH₄)2SO₄/L, 1 g MgSO₄7H₂O/L, 10 g yeast extract/L, 1 mL trace metal solution 1,000 X/L, and 50 μg kanamycin/mL. The trace metal solution 1X was prepared as follows: 2.29 mg AlCl3·6H₂O/L, 1.6 mg (CoCl2·6H₂O)/L, 41.21 mg H3BO3/L, 4.98 mg (MnSO₄·H₂O)/L, 0.73 mg (Na2MoO₄·2H₂O)/L, 13.7 mg (CuSO₄·H₂O)/L. Using a peristaltic pump, feed medium (265 g glucose/L and 50 g yeast extract/L) was added to the fed batch cultures at a constant flow rate of 0.7 mL/min when the pH had risen, because that was a signal that the initial carbon source had been depleted.

Culture Conditions

Shake-flask cultures were grown in 500 mL of LB medium and agitated at 200 rpm in a rotary shaking incubator for 6 h at 37 °C. Fed batch fermentations were aerated at 5 L/min and stirred at 400–700 rpm to maintain an oxygen saturation rate of 20%. The pH level was maintained at 7.0 ± 0.2 by automatic additions of 25% (v/v) NH3 and 85% (v/v) phosphoric acid. Foaming was controlled automatically by the addition of an antifoaming agent.

Dry Cell Weight Determination

To determine the dry-cell-weight, 1 mL aliquots of fermentation culture were centrifuged at 10,000 rpm for 10 min in preweighed plastic tubes. After careful removal of the supernatant, cells were resuspended in an equal volume of sterile purified water and centrifuged under the same conditions. The supernatant was decanted and the cell pellets were dried to constant weight at 105 °C.

Glucose and Total Protein Assay

Glucose concentration in the supernatant was determined by the dinitrosalicylic acid reducing-sugar assay.¹¹ Total protein concentration was measured using the Lowry method.

Plasmid DNA Isolation

Each replicate of the 10 mg bacterial cell pellet was resuspended in 300 μL of 50 mM Tris-HCl, 10 mM EDTA, 100 μg RNase A/mL, pH 8.0, and purified using the alkaline method.¹²

Gel Electrophoresis

Agarose gels (0.8%) were prepared in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0), containing 0.5 μg ethidium bromide/mL. Gels were run at 100 V (5 V/cm), for 1 h. The photographs were taken using a video camera (GelPrinter Plus, TDI, Madrid, Spain).

Analytical Method for Quantification

Absorbance of the DNA samples at 260 and 280 nm was measured using an HP spectrophotometer. The concentration of plasmid DNA was calculated from the A260 data. The purity of the samples was checked by the ratio of absorbance at 260 nm and 280 nm.

RESULTS AND DISCUSSION

Design of the High-Cell-Density Culture Medium

Media composition can dramatically affect plasmid quality and yield. A high-cell-density fermentation requires a balanced medium supplying adequate amounts of nutrients for energy, biomass, and cell maintenance, and commonly contains carbon and nitrogen sources, various salts, and trace metals. Taking these requirements into consideration, we designed a culture medium with a bacterial element composition similar to what has been reported in the literature.¹³

The results displayed in Figure 1 show that when cells were grown in this medium, cell growth was in the log phase from hour 1 to approximately hour 21, and in the stationary phase from approximately hour 22 to 25. Cell density then began to decrease at approximately hour 25 or 26.

Figure 1. Cell growth kinetics of recombinant E. coli DH10B transformed with pIDKE2 in the designed medium in a 5-L fed batch fermentation process

Carbon source exhaustion occurred after 5 h of batch growth, as indicated by the increase in pH (>7.2), which was caused by the consumption of the alternative carbon source and confirmed by a reducing-sugar assay. Starting precisely at that moment, a mixture of glucose and yeast extract was fed at a constant flow rate and glucose accumulation was observed only when feeding started.

During the initial batch period, a specific growth rate (μ) of 0.65 h-1 was achieved, whereas a lower specific growth rate of 0.07 h-1 was maintained during the fed batch period. It has been reported that low growth rates lead to a higher specific plasmid DNA yield than if cell growth is not inhibited. A reduced growth rate is critical for high quality, high yield fermentations for plasmid production because it allows for plasmid amplification and greater stability.¹⁴

The cellular yield achieved from the fermentation was 0.42 g dry cell/g of glucose, which is similar to what has been reported in the literature (0.5 g dry cell/g of glucose) for E. coli, based on the theory described by Carnes.¹⁴ On the other hand, the total protein concentration levels were high (around 7 g/L) after feeding started, because of the yeast extract, which contained high quantities of protein.

The highest cell mass (29±1.7 g dry cell/L), plasmid yield (154±2.8 mg plasmid DNA/L), and specific plasmid DNA yield (0.44±0.02 mg plasmid DNA/g dry cell weight) were obtained after 24 h of culture (Figure 1), so we suggest that culture be stopped after 24 h.

Taking into account that several studies have shown that the supercoiling linking number is an important factor to consider when processing pDNA for therapeutic use,⁸ we analyzed the percentage of supercoils at this time point in the culture (24 h) and found that the level was approximately 80% of the total plasmid DNA (Figure 2). Several articles have reported similar results because supercoiled pDNA is known to be more resistant to certain growth conditions than other isoforms.⁸

Figure 2. Plasmid DNA yield kinetics of recombinant E. coli DH10B transformed with pIDKE2 in the designed medium in a 5-L fed batch fermentation process

The behavior of E. coli (DH10B) in the fed batch fermentation was normal because the state of the system changed from one with a low initial cellular concentration (0.1 g dry cell weight/L) to a state with very high biomass and product concentrations.

Comparing Glucose and Glycerol as Carbon Sources

Glucose is a conventional carbon source because it is inexpensive and very efficiently metabolized. However, high glucose levels are known to cause undesirable acetate production as a result of metabolic overflow. The application of glycerol avoids the repression of intermediate metabolites and accumulations of inhibitive organic acids to some extent. Therefore, the effect of glycerol additions in culture and feed medium on plasmid DNA production in E. coli was also examined. The effects of glycerol and glucose as a carbon source in culture medium on plasmid production are shown in Figure 3.

Figure 3. Cell growth kinetics of recombinant E. coli DH10B transformed with pIDKE2 in a 5-L fed batch fermentation process. Effects of carbon source on plasmid production and cell growth.

The results showed that the highest cell mass (27.71±1.5 g dry cell weight/L) and specific plasmid yield (0.45±0.15 mg plasmid DNA/g dry cell weight) were obtained after 24 h of culture when glucose was chosen as the carbon source for the medium, whereas both cell mass and plasmid productivity were reduced when glycerol was used. Thus, compared to glycerol, glucose was the optimal carbon source when the initial concentration was 5 g glucose/L, and 265 g/L was added to the feed medium.

The feeding of nutrients, usually glucose, has been extensively researched and incorporates a range of approaches that span from simple to very elaborate, each presenting its own advantages and disadvantages.²

Effects of Nitrogen Source on Plasmid Production

The choice of nitrogen source and the determination of its concentration are critical to the optimization of plasmid production in recombinant cell fermentation. The bacterial requirements for nitrogen can be satisfied by inorganic or organic sources. In our work, yeast extract was used and the effects of different levels in the feed were tested.

Results (Figure 4) showed that the cell mass increased up to 28 g dry cell weight/L when the concentration of yeast extract was increased from 20 g/L to 50 g/L and the highest specific plasmid productivity (0.45 mg plasmid DNA g/L dry cell weight) was achieved. Therefore, from the viewpoint of the development of high-cell-density culture, an inorganic supply of nitrogen from complex components such as yeast extract was essential because it was more effective in supporting high plasmid yield.

Figure 4. Effects of yeast extract concentration on plasmid production and cell growth in a 5-L fed batch fermentation process

CONCLUSION

With this cell culture procedure, larger amounts of the plasmid pIDKE2 were obtained in DH10B cells. The results from this study may be beneficial for the development of techniques for fed batch fermentation of E. coli cells and for the efficient production of plasmid DNA for therapeutic use in humans.

Odalys Ruiz Hernández is the principal researcher, Mariela Pérez de la Iglesia, Saily Martínez Gómez, Karelia Macias Cosme, Michel Díaz Martinez, and Yanay Proenza Jimenez are researchers in the fermentation department and Jorge Valdés Hernández is the department head. Miladys Limonta Fernández is the principal researcher in the purification department. Marta Pupo Peña is a researcher and Dinorah Torres Idahody, PhD, is the department head, both in the analytical development department, and Eduardo Martínez, PhD, is the head of the development division, all at the Center for Genetic Engineering and Biotechnology, Havana, Cuba, odalys.ruiz@cigb.edu.cu +537.271.6022, ext: 5250.

REFERENCES

1. Prazeres D, Ferreira G, Monteiro G, Cooney C, Cabral J. Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy. Trends Biotechnol. 1999;17(4):169–74.

2. Prather KJ, Sagar S, Murphy J, Chartrain M. Industrial scale production of plasmid DNA for vaccine and gene therapy: Plasmid design, production and purification. Enzyme Microb Technol. 2003;33:865–83.

3. Schleef M, editor. Biotechnology. Volume 5a: Recombinant Proteins, Monoclonal antibodies and Therapeutic Genes. Hoboken, NJ: Wiley & Sons; 1999. p. 445–69.

4. Vogel FR, Sarver H. Nucleic acid vaccines. Clin Microbiol. 1995;8:406–10.

5. Robinson HL. DNA vaccines. Clin Microbiol Newslett. 2000;23:17–22.

6. Herweijer H, Wolf J. Progress and prospects: Naked DNA gene transfer and therapy. Gene therapy. 2003;10:453–8.

7. Chattergoon M, Boyer J, Weiner DB. Gene immunization: A new era in vaccines and immune therapeutics. FASEB. 1997 Aug;11(10):753–63.

8. O'Kennedy R, Ward J, Keshavarz E. Effects of fermentation strategy on the characteristics of plasmid DNA production. Biotecnol Appl Biochem. 2003 Feb;37 (Pt 1):83–90.

9. Kim B, Shuler M. Analysis of pBR322 replication kinetic and its dependency on growth rate. Biotechnol Bioeng. 1990;36:233–42.

10. Xu Z, LiuG, Cen P, Wong W. Factors influencing secretive production of human epidermal growth factor (hEGF) with recombinant E. coli K12. Bioprocess Biosyst Eng. 2000;23:669–74.

11. Miller G. Anal. Chem. 1959 31:426–8.

12. Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979;7:1513–23.

13. Shiloach J, Fass R. Growing E. coli to high cell density—A historical perspective on method development. Biotechnol Adv. 2005;23:345–57.

14. Carnes A. Fermentation design for the manufacture of therapeutic plasmid DNA. BioProcess Int. 2005 Oct;3(9):36–44.