Growth Medium Selection and Its Economic Impact on

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medium and using LB as a base scenario (100 medium cost [MC] units/mg pDNA), the ... PDMR medium yielded pDNA at a cost of only 27 MC units/mg pDNA.
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 104, No. 6, 490–497. 2007 DOI: 10.1263/jbb.104.490

© 2007, The Society for Biotechnology, Japan

Growth Medium Selection and Its Economic Impact on Plasmid DNA Production Michael K. Danquah1* and Gareth M. Forde1 Bioengineering Laboratory, Department of Chemical Engineering, Monash University, Clayton campus, Wellington Rd., Victoria 3800, Australia1 Received 22 January 2007/Accepted 27 September 2007

Current developments in gene medicine and vaccination studies are utilizing plasmid DNA (pDNA) as the vector. For this reason, there has been an increasing trend towards larger and larger doses of pDNA utilized in human trials: from 100–1000 µg in 2002 to 500–5000 µg in 2005. The increasing demand of pDNA has created the need to revolutionalize current production levels under optimum economy. In this work, different standard media (LB, TB and SOC) for culturing recombinant Escherichia coli DH5α harbouring pUC19 were compared to a medium optimised for pDNA production. Lab scale fermentations using the standard media showed that the highest pDNA volumetric and specific yields were for TB (11.4 µg/ml and 6.3 µg/mg dry cell mass respectively) and the lowest was for LB (2.8 µg/ml and 3.3 µg/mg dry cell mass respectively). A fourth medium, PDMR, designed by modifying a stoichiometrically-formulated medium with an optimised carbon source concentration and carbon to nitrogen ratio displayed pDNA volumetric and specific yields of 23.8 µg/ml and 11.2 µg/mg dry cell mass respectively. However, it is the economic advantages of the optimised medium that makes it so attractive. Keeping all variables constant except medium and using LB as a base scenario (100 medium cost [MC] units/mg pDNA), the optimised PDMR medium yielded pDNA at a cost of only 27 MC units/mg pDNA. These results show that greater amounts of pDNA can be obtained more economically with minimal extra effort simply by using a medium optimised for pDNA production. [Key words: plasmid DNA, cultivation medium, fermentation]

ultimately full-scale production under optimum economy. Plasmid DNA is produced from a fermentation process and the success of this fermentation process hinges on the interactions between the host organism harbouring the recombinant plasmid vector and the growth environment (8). A major advantage of fermentation is that conditions that influence cell growth, plasmid yield, quality and stability can be examined and controlled. These include media composition, temperature, pH, dissolved oxygen and build-up of waste metabolites. Plasmid DNA vaccine doses are increasing more than the amounts currently produced by fermentations (2). The cultivation medium formulation dramatically affects the performance and nature of microbial processes. This implies that, the type and source of nutrients available in the growth medium can have a profound effect on the fermentation yields (1, 9). Media containing yeast extract and hydrolysed protein are often used because they are relatively simple to prepare and generally lead to high cell densities. Meat extracts are also rich sources of nutrients for fermentation, but there is the risk of contamination with animal viruses (1). In addition to complex nutrients glucose, glycerol or other sugars are included as a source of carbon and energy. Nitrogen is usually provided in inorganic form such as ammonium salts (9). Trace metals and vitamins may also contribute to cell growth and plasmid yield. Defined media pre-

Recombinant DNA technology and the sequencing of the human genome have led to revolutionary discoveries especially in the fields of gene therapy and nucleic acid vaccines (1, 2). Plasmid DNA has acquired appreciable interest due to its attractive potential application in gene therapy and DNA vaccines applications (3). Gene therapy processes involve the introduction of one or more functional and specific genes into a human recipient to repair certain genetic defects and aberrations. A pDNA vaccine can be developed from a pathogen’s genes to provide immunity against diseases (4, 5). Plasmid DNA vaccines allow the foreign genes to be expressed transiently in transfected cells, mimicking intracellular pathogenic infection and inducing humoral and cellular immune responses (6, 7). Plasmid DNA vaccines offer a new opportunity to immunize with materials that are mainly gene-based and are expressed by the cells of the recipient. This gives greater control over the entire immunization process (2). Considerable attention has been given to the potential of pDNA vaccines to mitigate and prevent a number of infections but substantially less examination has been given to the practical challenges of producing large quantities of pDNA for therapeutic use in humans, for both clinical studies and * Corresponding author. e-mail: [email protected] phone: +61-399053440 fax: +61-399055686 490

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pared from purified components instead of complex biological mixtures may be more reproducible since different preparations of complex substrates may vary somewhat in composition of vital elements. However, defined media usually require more components, are more difficult to prepare, and often lead to lower cell densities than can be achieved with complex media (1). Hence there is a challenging multivariable trade-off or decision to be made regarding growth of biomass, plasmid yield, quality, stability and cost in the selection of medium for pDNA production. Escherichia coli is a micro-organism that grows in both rich complex organic media and salt-based chemically defined media as long as carbon source is present (2). Besides an energy source, it requires nutrients for the biosynthesis of cellular matter, formation of products and maintenance so the content of the medium must supply the nutrients needed to accomplish this. The vital chemical elements needed for the cultivation of E. coli are hydrogen, carbon, nitrogen, oxygen, sodium, magnesium, phosphorus, potassium and calcium. These have specific functions during the bioactivity of cell growth and plasmid yield. Physiologically, hydrogen and oxygen form the basis of cellular water and with carbon as the main constituent of organic cell materials. Sodium, potassium, magnesium and calcium are cellular cations and cofactors for some enzymes. Phosphorous constitutes phospholipids, coenzymes and nucleotides in nucleic acids (9, 10). The type and concentration of these elemental ingredients used in cultivation medium determines the amount of biomass produced, as well as plasmid volumetric yield and specific yield. It is also likely that medium composition will directly bear on the physiology of the microorganisms by influencing their intricate regulatory systems, and therefore will control plasmid copy number (2). Plasmids DNA for gene therapy or vaccination harbouring genes are very large molecules in comparison to proteins. Knowledge about culture media and conditions for the fermentation of recombinant E. coli has been obtained on the basis of studies, which optimised the expression of proteins (11–13). However, nutrient conditions for optimisation of pDNA production in E. coli could be significantly different from those of protein production. Replication is the only process required when pDNA is the final product. Transcription and translation as in the case of proteins are generally undesirable during pDNA production in bacterial cells (4). Media suitable for E. coli can be purchased or their formulations can be obtained from the literature. Compositions of complex and defined media are extensively discussed in many publications (14–17). Due to the simplicity of use associated with offthe-shelf cultivation media, a number of laboratory-scale pDNA vaccine production schemes rely on the use of unoptimised small-scale processes employing commercially available complex formulations (18, 19). Although many works on pDNA production in E. coli by fermentation are reported using different media, reports on the experimental examination of comparative effect of different media on cell growth and pDNA yield kinetics as well as economic consideration of medium formulation are limited. This limitation makes cultivation medium selection more of a trial and error process without any medium cost per pDNA yield analysis based on previous fermentation

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works in the literature. This constitutes generally to the low pDNA yield that is encountered even for expensive medium formulations and high copy number plasmids. Studies on pDNA yield kinetics of a particular medium dictates when fermentation can be halted for maximum yield if it is employed rather than the normal random stoppage which constantly produces small amounts of pDNA. This work is geared toward investigating the effects of different culture media on biomass growth as well as yield and purity of pDNA. An economic comparison between different media (cost of medium per mg of pDNA produced), generally not considered in the literature to date, is presented here. Economic modification of stoichiometric medium and improvement of standard and stoichiometric media performances on cell growth and plasmid yield based on glucose addition and optimisation of carbon to nitrogen ratio and cost analysis are also reported. Economic evaluation of growth medium for large scale pDNA production is discussed. MATERIALS AND METHODS Materials Maximum efficiency E. coli DH5α (EndA–), pUC19 plasmid (0.01 µg/l) were purchased from Invitrogen (Victoria, Australia), tryptone (Difco, NJ, USA), yeast extract (Difco), NH4Cl (Sigma-Aldrich, NSW, Australia), K2HPO4 (Merck, Whitehouse Station, NJ, USA), ethidium bromide (Sigma-Aldrich), NaOH (Sigma-Aldrich), KH2PO4 (Merck), HCl (Sigma-Aldrich), glycerol (Amresco, OH, USA), NaCl (Amresco), KCl (Sigma-Aldrich), MgCl2 (Univar, WA, USA), MgSO4 (Sigma-Aldrich), glucose (Merck), propylene glycol (Sigma-Aldrich) and Bradford reagent (SigmaAldrich). Media Preparation Compositions of LB, TB and SOC media used for shake flask fermentation are as shown in Table 1. Wang et al. (4) reported the rational design of a defined medium optimised for pDNA production by E. coli strain JM109 harbouring the pcDNA3S plasmid using a stoichiometric approach. They considered the effect of glucose, amino acid and nucleosides concentrations on cell growth and pDNA yields. Maximum growth yield coefficient of glucose in the synthesis of cell biomass θglc was obtained as 0.40 g DCW/g glucose. After identifying six key amino acids (Asp, Glu, Gly, His, Leu, Try) to add to a glucose-basal salt medium, the designed formulation supported higher pDNA volumetric and specific yields when compared with those routinely achieved in the complex LB medium. When the defined medium was supplemented with nucleosides (adenosine, guanosine, cytidine, and thymidine), the pDNA volumetric and specific yields were further enhanced. The final concentration of nutrients in the medium (MW1) obtained from the stoichiometric model were 1.240 g/l asparate, 1.152 g/l glutamine, 0.378 g/l glycine, 0.111 g/l histidine, 0.693 g/l leucine, 0.03 g/l tryptophan, 0.096 g/l adenosine, 0.099 g/l guanosine, 0.146 g/l cytidine, 0.156 g/l thymidine, 10 g/l glucose, 12.8 g/l Na2HPO4 ⋅ 7H2O, 3 g/l KH2PO4, 0.5 g/l NH4Cl, 0.24 g/l MgSO4, and 0.004 g/l thiamine. This medium gave a high plasmid yield of 60 mg/l in batch fermentation of E. coli JM109 pcDDA3S. TABLE 1. Composition of LB, TB and SOC media prepared at room temperature Medium LB TB SOC

Composition (l–1) 10 g tryptone, 5 g yeast extract, 5 g NaCl 12 g tryptone, 24 g yeast extract, 9.4 g K2HPO4, 2.2 g KH2PO4, 4 ml glycerol 20 g tryptone, 5 g yeast extract, 0.6 g NaCl, 0.2 g KCl, 0.95 g MgCl2, 1.2 g MgSO4, 3.6 g glucose

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TABLE 2. Composition of medium PDM prepared at room temperature Composition Yeast extract Tryptone Glucose Na2HPO4 ⋅ 7H2O KH2PO4 NH4Cl MgSO4

Amount (g/l) 4.41 7.93 10.00 12.80 3.00 0.50 0.24

Following a critical economic consideration of medium MW1 in both analytical and large scale production of pDNA on the grounds of high cost of amino acids, nucleosides and thiamine, this paper reports the formulation of an optimised and economically viable medium PDM (plasmid DNA medium) for pDNA production via fermentation. Yeast extract and tryptone were identified to contain the required amino acids (Asp, Glu, Gly, His, Leu, Try), nucleosides (adenosine, guanosine, cytidine and thymidine) and thiamine for cell growth and pDNA yield as in medium MW1 whilst being economically preferable. Tryptone contains approximately 35% amino acids (Asp, Glu, Gly, His, Leu, Try), and 0.33 µg/g thiamine. Yeast extract contains approximately 19% amino acids (Asp, Glu, Gly, His, Leu, Try) and 3.2 µg/gm thiamine. Tryptone and yeast extract contain 0.75% and 0.29% respectively of phosphorus which support the building of nucleotides (Difco & BBL manual, 2003). Calculated amounts of yeast extract and tryptone were used for medium PDM (Table 2) based on the aforementioned compositions to make up the content of amino acid, nucleoside and thiamine as for medium MW1. Cell transformation Maximum efficiency E. coli, DH5α (EndA–), cells were transformed with pUC19 plasmid as per the manufacturer’s instructions (Invitrogen). Cell lines were created (LB-glycerol), frozen in a dry ice/ethanol bath for 5 min then stored at −75°C (Nuaire, Tokyo) until further use. Shake flask fermentation process A single bacterial colony carrying the pUC19 plasmid (E. coli DH5α-pUC19) was picked from the LB-agar-ampicillin plate and subcultured with 100 ml of LB culture containing an appropriate amount of ampicillin (100 µg/ml) at 37°C overnight under 200 rpm shaking. Subsequently, 2 ml of the culture was inoculated into each of 200 ml LB, TB, SOC and PDM containing 100 µg/ml of ampicillin. Fermentation was allowed overnight at 37°C and 200 rpm shaking. Fermentation process was halted after 15 h. Fermentation monitoring Two milliliter sample of each culture was pipetted aseptically after every 30 min and part was used for optical density determination (OD600) using a spectrophotometer (Shimadzu, Kyoto). The remaining was spun in a centrifuge (Heraeus, Victoria, Australia) at 10,000×g for 5 min at 4°C. The supernant liquid was gently decanted and the cell resuspended in PBS buffer and stored in −75°C freezer. Cells were later lysed, purified for pDNA and analysed for quantity and purity. Batch fermentation process One single bacterial colony carrying the plasmid was picked and subcultured with 1 l of LB culture containing an appropriate amount of ampicillin at 37°C overnight under 200 rpm shaking. Subsequently, 500 ml of the culture was inoculated into a 20 l fermentor (New Brunswick Scientific, Edison, NJ, USA) vessel containing 15 l of PDMR medium (PDM with optimum C/N ratio) and 100 µg/ml of ampicillin. The temperature was set at 37°C and the dissolved oxygen (DO) value was maintained at 30% by the proportional integral derivative (PID) controller, which changed the speed of agitation to maintain the set DO value. The pH was maintained at 7.0 by the addition of 4 M NaOH and 1 M HCl. The inflow sparge air at a flow rate of 7.2 slpm was set at 20 psia and foaming was checked by using poly-

propylene glycol as antifoam. Culture sampling was performed after every 30 min to monitor biomass growth as well as pDNA yield kinetics. The cultivation was terminated 15 h after inoculation of fermentor. The culture broth was harvested and then concentrated by ultrafiltration. The obtained cell paste was packaged and stored at −75°C prior to lysis. Dry cell concentration determination Ten milliliter aliquots of cell culture with known OD600 were spun at 3700×g in a centrifuge for 10 min at 4°C. Supernant was decanted and the cell pellets were washed with equal volume of sterile water. Cell were transferred to a pre-weighed glass plate and dried over night to a constant weight at 105°C. Cell lysis and plasmid DNA purification Plasmid DNA purification from bacterial cell was performed with Wizard Plus SV Minipreps (Promega) according to the manufacturer’s instructions. Briefly, cells were alkaline lysed, clarified, plasmid was column purified and eluted prior to quantification. Plasmid DNA analysis Quantity and purity of pDNA samples from LB, TB, SOC and PDM media were determined from optical density analysis, OD260 and OD280. Nature and size of plasmid DNA were determined by ethidium bromide agarose gel electrophoresis using a 1 kbp DNA ladder. Gel was made up in ×50 dilution of TAE buffer (242 g of Tris base, 57.1 ml acetic acid, 9.305 g of EDTA), stained with 3 µg/ml EtBr and run at 66 V for 2 h. Gel was scanned with gel analyser (Bio-Rad, Segrate, Italy). Protein quantification analysis was performed using Bradford assay according to the manufacturer’s instructions.

RESULTS AND DISCUSSION Transformation efficiency A transformation efficiency of 5 ×108 CFU/µg pUC19 obtained shows that appreciable amount of bacterial cells (E. coli DH5α) were transformed with pUC19 and this reveals favourable cell viability. Biomass growth kinetics: shake flask fermentation Optical density values at 600 nm measured for samples from each of the bacterial culture, LB, TB, SOC and PDM at 30 min interval were converted to dry cell concentration (mg/ml). Different biomass growth profiles of E. coli DH5α pUC19 were portrayed by the different media (Fig. 1A). The growth kinetics of the cells in the different media was about the same in the first 3 h and this similarity continued for TB, SOC and PDM up to the 5th hour. Cell growth in LB medium started stabilizing after 10 h reaching a final cell concentration of 0.85 mg/ml. Significant variation existed in the cell densities for the different media after 10 h and persisted until fermentation was halted after 15 h. This variation is attributed to the difference in the amount of carbon source present in the different media since biomass growth is boosted mainly by the presence of carbon source. The slow biomass growth in SOC after 9 h is due to the exhaustion of its glucose (carbon source) content. The final dry cell concentrations for PDM and TB were 1.94 mg/ml and 1.80 mg/ml respectively with the expectation of further increment. There is some level of similarity in PDM and TB media support for biomass growth considering their growth profiles and final cell optical densities. Both TB and PDM attained a maximum growth rate of 0.22 h–1. Plasmid DNA yield kinetics: shake flask fermentation The concentration of pDNA was analysed spectrophotometrically via absorbance at 260 nm. The optical density of 1.0 measured at 260 nm with light path of 1 cm represents

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FIG. 1. (A) Dry cell concentration profiles obtained from OD600 measurements for the growth of E. coli DH5α pUC19 in LB, TB, SOC and PDM media in shake flask fermentation at 37°C for 15 h under 200 rpm shaking. Samples were collected at 30 min intervals (n = 3). (B) Volumetric yield characteristic of pDNA purified from LB, TB, SOC and PDM media. Samples were collected at 30 min intervals purified and analysed spectrophometrically at OD260 under room temperature. 1 OD260 unit = 50 µg/ml double stranded DNA (n = 3). (C) Specific yield characteristic of pDNA purified from LB, TB, SOC and PDM media. Samples were collected at 30 min intervals. Specific yield is the ratio of the volumetric yield to the cell concentration. Symbols: rhombuses, LB; squares, TB; triangles, SOC; crosses, PDM.

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50 mg/l of double stranded DNA. Analysis of samples of pDNA purified from LB, TB, SOC and PDM bacterial cell cultures showed different plasmid yield kinetics for the different media (Fig. 1B). After 3 h of near negligible pDNA production, the volumetric yield of pDNA increased over the final 12 h reaching 2.8 µg/ml, 11.4 µg/ml, 6.4 µg/ml and 14.6 µg/ml for LB, TB, SOC and PDM media respectively. There is a clear distinction between the pDNA volumetric yield characteristics of PDM and TB media; this may be attributed to the stoichiometrically designed PDM containing optimised amino acid, nucleoside and thiamine concentrations for pDNA production. Figure 1C shows a similar trend for the specific yield of pDNA for the different media. There exists some level of similarity in the progression of pDNA specific yield for the LB, TB and SOC, most notably a kink in the specific yield profile between 4–6 h. This phenomenon is ascribed to the fact that in the exponential phase of a batch profile, growth rate of biomass is maximal and it overtakes the replication rate of pDNA thereby causing a decrease in specific yield. This scenario was not observed in PDM profile as the rate of pDNA volumetric yield matches the increase in biomass during the exponential growth phase. Absorption measurements taken at wavelengths of 260 nm and 280 nm were used to determine the purity of double stranded DNA based on the ratio OD260/OD280 which is expected to be within 1.7–1.9 to indicate pure double stranded DNA. A low OD260/OD280 ratio indicates protein contamination whilst a high OD260/OD280 shows possible RNA or residual organics contamination. The final purity of DNA from the PDM medium was the highest and that from the LB was the lowest (Fig. 2A). Ethidium bromide agarose gel electrophoresis (Fig. 2B) shows no RNA or gDNA contamination and the purification protocol ensures that organics are removed from the system before elution; hence the high OD260/OD280 ratio for PDM indicates the presence of supercoiled pDNA. Protein quantification analysis using Bradford assay shows protein concentration of ~0.4%, which is keeping with the regulatory standard of < 1%. The highest specific yield of plasmid obtained from the PDM medium has an advantageous effect on plasmid purity since the quantity of impurities existing with the plasmid per unit mass of cell is minimal. Effect of glucose on LB performance LB performance was modified by the gradual addition of glucose (LB+ x% where x is the % w/v of glucose as carbon source) and subjected to the same condition of shake flask fermentation of E. coli DH5α-pUC19. The growth of biomass was greatly influenced by the right proportion of carbon source. Much of the carbon source taken up by E. coli enters the pathways of energy yielding (ATP) metabolism and is eventually secreted from the cell as CO2 (the major product of energy yielding metabolism). According to Fig. 3, the maximum dry cell concentration (1.03 mg/ml) was obtained at a glucose concentration of 4% w/v. The decrease in dry cell density at glucose concentrations above 4% w/v is as a result of the excess glucose concentration in the growth medium which posses an extra metabolic burden to the cells, thereby retarding their proliferation rate and hence the dry cell concentration. The addition of glucose also boosted both pDNA volu-

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FIG. 2. (A) Plasmid DNA specific yield and purity obtained from LB, TB, SOC and PDM. Purity was measured by the ratio of OD260 to OD280. PDM medium resulted with the highest plasmid purity due to the high specific yield of pDNA. Black bars, Final specific yield; gray bars, OD260/OD280 (B) Results from ethidium bromide agarose gel electrophoresis of pDNA samples from LB, TB, SOC and PDM media. Analysis was done using 1% agarose in TAE ×1 buffer, 5 µg/ml EtBr at 66 V. Lane M is 1 kbp DNA ladder, lanes 1, 2, 3 and 4 represent the pDNA samples from PDM, TB, SOC and LB, respectively.

FIG. 3. Dry cell concentration of E. coli DH5α pUC19 in LB + x% where x is the percentage amount of glucose as carbon source (0– 12%). Shake flask fermentation was performed at 37°C for 15 h under 200 rpm shaking (n = 3).

metric and specific yields (Fig. 4) since glucose is needed for the synthesis of nucleotides in pDNA. A maximum pDNA volumetric and specific yield of 3.9 µg/ml and 3.80 µg/mg dry cell mass respectively at 4% w/v of glucose were obtained. Effect of C/N ratio on LB and PDM performances The ratio of glucose to NH4Cl in LB medium was adjusted between 1: 1 and 8:1 by altering the concentration of NH4Cl. The result (Fig. 5) showed a tremendous influence of C/N ratio on plasmid specific yield. The maximum plasmid specific yield was obtained C/N = 3 resulting in a specific yield of over twice that of the un-optimised LB medium. The theoretical C/N optimum value was found to be in the range 2.5– 3.2. O’Kennedy et al. (20) reported an optimum C/N ratio of 2.78 for SDCAS medium. C/N ratio in the PDM medium was varied between the optimum range of 2.4–3.2 by altering the concentration of NH4Cl. The highest pDNA specific yield (11.17 µg/mg dry

FIG. 4. Plasmid DNA volumetric and specific yield profiles for LB + x% where x is the percentage amount of glucose as carbon source. Shake flask fermentation was performed at 37°C for 15 h under 200 rpm shaking (n = 3). Symbols: rhombuses, volumetric yield; squares, specific yield.

cell mass) occurred at C/N ratio of 2.8 corresponding to a biomass yield of 2.13 mg/ml. The C/N-optimised PDM medium is referred to as PDMR. Effect of phosphate concentration on plasmid production The fundamental building blocks of a pDNA molecule are nucleotides. These nucleotides are made of a phosphate group, a sugar ring and a nitrogen base so the presence of phosphate in a growth medium can have an influence on the synthesis of nucleotides thereby affecting pDNA yield. LB and SOC have approximately negligible phosphate concentration except for the little amount present in yeast extract and tryptone which amounts to only 0.04% and 0.3% respectively. The total quantity of phosphate in LB and SOC media were evaluated to be 0.09 g/l and 0.18 g/l respectively (Fig. 6). The difference in the specific yield of LB and SOC is partially attributed to the presence of extra phos-

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FIG. 5. Effect of C/N ratio on pDNA production from LB and PDM medium. C/N ratios were achieved by adjusting concentration of NH4Cl. Shake flask fermentation was performed at 37°C for 15 h under 200 rpm shaking. Symbols: rhombuses, LB; squares, PDM.

FIG. 7. Dry cell concentration profiles obtained from OD600 measurements for the growth of Escherichia coli DH5α pUC19 in PDMR medium. Batch fermentation was carried out in a 20 l fermentor at 37°C for 15 h. Samples were collected at 30 min intervals (n = 3).

FIG. 6. Dependency of pDNA specific yield on phosphate concentration of different growth media LB, TB, SOC and PDMR. Black bars, Specific yield; gray bars, total phosphate concentration.

FIG. 8. Plasmid DNA volumetric and specific yield profiles for PDMR medium. Batch fermentation was carried out in a 20 l fermentor at 37°C for 15 h. Samples were collected at 30 min intervals (n =3). Symbols: squares, volumetric yield; triangles, specific yield.

phate concentration in SOC and the presence of other essential nutrients such as carbon source and trace metals. TB and PDMR showed higher pDNA specific yields than LB and SOC; possibly due to the availability of extra phosphates groups. However, the pDNA specific yield from TB is found to be lower than that of PDMR though it contains a higher phosphate concentration. TB as a commercially available medium contains nutrients which are not perhaps optimized solely for pDNA replication but for cell proliferation and other biomolecules synthesis such as proteins. On the other hand, PDMR contains the right nutrients (P:C:N) whose quantities are stoichiometrically evaluated and tuned in the right proportion for plasmid replication, hence the highest pDNA specific yield. Performance of PDMR employed in batch fermentation Batch cultivation of E. coli DH5α-pUC 19 using PDMR medium showed a massive increase in biomass yield as well as plasmid volumetric and specific yields. After an initial lag of 3 h the biomass yield increased to 0.26 mg/ml and to 4.55 mg/ml after the next 12 h of cultivation (Fig. 7). Biomass yield increased continuously throughout the entire cultiva-

tion period with the expectation of further increment. This continuous increase in biomass is obviously due to the available amount of carbon source present in PDMR medium for cell growth. Glucose uptake rate and metabolism by cell were enhanced due to oxygen availability resulting from sparged air forced into the system. The maximum growth rate attained during cultivation was 0.45 h–1. As shown in Fig. 8, there was a general correspondence between plasmid volumetric yield and biomass growth within the first 12 h of cultivation. After the initial 3 h of nearly zero plasmid production, the volumetric yield of plasmid increased to 1.1 µg/ml and to a maximum of 62.6 µg/ml after 9 h. However, plasmid volumetric yield started declining from the 12th hour to a final value of 35.9 µg/ml. This is possibly due to the use of glucose as a carbon source which usually increases the maximum specific growth rate during the exponential phase of batch fermentation (where rate of metabolism is improved) and maximizes acetate production which is detrimental to pDNA replication. An alternative carbon source will be glycerol (21). Similar trend was observed for

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the pDNA specific yield. The maximum pDNA specific yield of 18.07 µg/mg obtained from the batch cultivation of PDMR compared to those routinely achieved reveals the potential of PDMR for use in large scale pDNA production with minimum cultivation period. Economic evaluation The cost inventory for a typical batch shake flask fermentation process for pDNA production encompasses of all materials and equipment for the actual fermentation work as well as downstream purification. The medium is a key critical input as it has direct impact both on the pDNA yield (as shown earlier) and the cost. Medium selection is often based on following previously developed protocols or, at best, trial and error without any cost per yield of pDNA analysis prior to fermentation. This is the main reason for the low pDNA yields attained even for expensive medium formulations and high copy number plasmids. A medium cost per pDNA yield analysis was performed on LB, TB, SOC, PDM and PDMR using standard LB medium as a base scenario of 100 medium cost units, where 100 MC units ~ US$ 0.58. The cost of medium was calculated based on the following relation. Ck = ∑αi,k mi,k + ∑ βj,kνj,k i=1

(1)

j =1

Where Ck is the total cost of medium k, αi,k is the cost per unit weight of components i existing in medium k, mi,k is the weight of component i present in medium k, βj,k is the cost per unit volume of components j existing in medium k and νj,k is the volume of component j present in medium k. From the results obtained from the economic study as shown in Fig. 9, the cost of medium per mg pDNA from PDM medium was 29 MC units and that of TB was 65 MC units. C/N ratio optimised PDM medium (PDMR) gave the highest plasmid yield at 27 MC units/mg pDNA (Table 3). PDMR medium increased pDNA yield to an extent that it reduced the cost of medium per mg pDNA. Application of PDMR in batch fermentations displayed an average volumetric yield of 62.6 mg/l which is slightly higher than that of the original MW1 in Wang et al. (~60 mg/l) (4). Aside this raw volumetric yield comparison, the cost per yield analysis which is the main thrust of this paper, favours outstandingly the PDMR medium (7 medium cost (MC)/mg pDNA) more than the original MW1 (35.2 MC/mg pDNA). However, costs of compressed air/oxygen and utilities can not be omitted in a complete cost evaluation of the batch run. Considering this eco-

FIG. 9. Medium cost per mg plasmid DNA from LB, TB, SOC, PDM and PDMR. The result shows medium cost per plasmid DNA yield advantage of PDMR. 100 units = US$ 0.58 for standard LB medium. Shake flask fermentation was performed at 37°C for 15 h under 200 rpm shaking (n = 3).

nomic evaluation, there is a great cost per yield advantage of using PDMR medium for small scale batch production, which shows promise for larger scale production of pDNA. Sedegah et al. (22) reported that pDNA vaccine doses used for human trials are commonly 500–5000 µg. For medium alone, this corresponds to 8.5–85 MC units (~US$ 0.05–0.50) when PDMR is used compared to 50–500 MC units (~US$ 0.29–2.90) for LB medium. Growth medium selection and economics for large-scale production of pDNA From a manufacturing perspective, large scale production of pDNA starts with high throughput fermentation. Medium formulation is a fundamental and challenging consideration in large scale fermentation as it requires a decision to be made between the cost of nutrients, plasmid yield and quality. Considerations that must be put forward in developing a medium for large scale pDNA production are: (i) The optimum nutrient requirements of the host organism; (ii) Comparison of nutrient requirements to the composition of available industrial and off-the-shelf nutrients. If not compatible, plan a formulation; (iii) Nutrient properties with respect to storage, handling, sterilization, processing and pDNA purification; and (iv) cost inventory of the ingredients. Computation of the cost of the medium for large scale production of pDNA involves the recognition of all cost re-

TABLE 3. Summary of pDNA yield and economic study results obtained for the various media investigated in shake flask and batch fermentation Dry cell concn pDNA vol. yield pDNA specific yield Medium cost/mg pDNA (g/l) (mg/l) (mg/g) (units) LB 0.85 2.8 3.29 100 LB (C optimised) 1.03 3.9 3.80 135 LB (C/N optimised) 1.18 6.1 5.17 117 TB 1.08 11.4 6.33 65 SOC 1.31 6.4 4.86 81 PDM 1.94 14.6 7.53 29 PDMR 2.13 23.8 11.17 27 PDMR (batch) (maximum yield) 3.66 62.6 17.10 7 PDMR (batch) (final yield) 4.55 35.9 7.89 12 PDMR medium is obtained from C/N optimisation of PDM. Data shown are the average values of three replicates (n=3).

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MEDIUM SELECTION AND ECONOMICS FOR pDNA PRODUCTION

lating to the medium. Thus, in addition to the main purchase price or cost of formulation, cost of material handling and storage, labour and analytical requirements must be estimated. Medium storage and handling depends on the type and nature of the nutrients. Liquid nutrients require larger storage capacities than slurry or concentrated nutrients. However, most of the nutrients required for bacteria fermentation for pDNA production exist in powder forms; hence less storage capacity is required. The rheological properties of the medium may affect the cost of operations such as mixing, aeration and temperature control. The concentration of the pDNA influences its recovery and purification costs. Productivity per unit process capacity helps determine the amount of capital, labour, and indirect costs assignable to pDNA. Reducing manufacturing costs by replacing nutrients with cheaper ones may not be the solution. A better approach would be to explore how the impact of a change in nutrient would affect the product yield and purity in addition to the cost analysis. ACKNOWLEDGMENTS Funding for this research was kindly provided by the Victorian Endowment for Science, Knowledge and Innovation (VESKI) and via the Monash University Early Career Researcher Grant Scheme.

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