(553a) Process Engineering Aspects of Plasmid-Based Biopharmaceuticals Production: Tackling the Threatening Vaccine Shortages to Prevent Global Pandemics

Infectious diseases such as SARS, influenza and bird flu have the potential to cause global pandemics; a key intervention will be vaccination. Hence, it is imperative to have in place the capacity to create vaccines against new diseases in the shortest time possible. In 2004, The Institute of Medicine asserted that the world is tottering on the verge of a colossal influenza outbreak. The institute stated that, inadequate production system for influenza vaccines is a major obstruction in the preparation towards influenza outbreaks. Because of production issues, the vaccine industry is facing financial and technological bottlenecks: In October 2004, the FDA was caught off guard by the shortage of flu vaccine, caused by a contamination in a US-based plant (Chiron Corporation), one of the only two suppliers of US flu vaccine. Due to difficulties in production and long processing times, the bulk of the world's vaccine production comes from very small number of companies compared to the number of companies producing drugs.

Conventional vaccines are made of attenuated or modified forms of viruses. Relatively high and continuous doses are administered when a non-viable vaccine is used and the overall protective immunity obtained is ephemeral. The safety concerns of viral vaccines have propelled interest in creating a viable replacement that would be more effective and safer to use.

Nucleic acid molecules are heralding a new generation of reverse engineered biopharmaceuticals. One example is plasmid DNA (pDNA) which has an excellent safety profile as it is free from safety concerns associated with viral vectors, displays no toxicity and is simpler to develop. Hence pDNA is considered a viable alternative to viral vectors. Reverse designed pDNA vaccines have the potential to revolutionalize production time and methods, aside being a safer way to invoke immune responses. The utilization of pDNA as a vector for the introduction of genes into mammalian cells via traditional immuno-response methods is widely reported. The increasing number of pre-clinical and clinical trials utilizing pDNA has resulted in pressure to make more in less time. 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 to meet the current rising demand. Plasmid DNA is mainly produced from fermentation processes and the success of this fermentation process hinges on the interactions between the host organism harboring the recombinant plasmid vector and the growth environment. 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. The growth medium formulation dramatically affects the performance and productivity of microbial processes, especially bacterial fermentation. Media containing yeast extract and hydrolyzed protein are often used because they are relatively simple to prepare and generates high cell densities. Meat extracts are also rich sources of nutrients for fermentation, but there is the risk of contamination with animal viruses. Commercially available growth media are usually employed for the fermentation of E. coli for pDNA production but most of these on-sale media are directed toward cell proliferation and/or protein expression and not pDNA replication. Due to the simplicity of use associated with these off-the-shelf media, a number of lab-scale pDNA vaccine production schemes rely on the use of un-optimized small-scale processes employing these complex formulations. Studies on the quantitative investigation of comparative effects 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 cost and yield analysis prior to fermentation. 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 can also dictate when fermentation can be halted for optimum performance rather than the normal random stoppage which constantly produces small quantities of pDNA.

Molecular biologists have developed efficient lab-scale protocols for pDNA purification which employ operations such as ultracentrifugation and solvent extraction. Although routinely used for obtaining pDNA for research applications, these protocols are not scalable or use reagents such as phenol, ethidium bromide, RNase or CsCl that are expensive or not compatible with regulatory guidelines. Further disadvantages include long processing times, limited capacity, low recovery and high cost per dose. The creation of a commercially viable purification process for pDNA requires a scalable technique employing optimized stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian and bovine derived enzymes in a minimum number of processing steps. It is in this light that a chromatographic technique for rapid pDNA purification is essential.

The isolation and purification of pDNA is hampered by the low performance of conventional chromatographic supports with a small particle pore diameter. Most of these chromatographic supports are made for the high adsorption capacity of proteins with size less than 10 nm. In columns packed with such supports, large molecules such as pDNA with sizes greater than 100 nm adsorb predominantly at the particle outer surface. Consequently capacities are in the order of tenths of mg pDNA/mL support compared to hundreds of mg/mL for proteins.

A monolith is a continuous phase consisting of a single piece of a highly porous organic or inorganic solid material. The most important feature of this support is that all the mobile phase is forced to flow through the large pores of the monolith. As a consequence, mass transport is enhanced by convection; dramatically reducing the long diffusion time required by particle based chromatographic supports. Therefore, the chromatographic separation process on monoliths is practically not diffusion-limited. The large pores of these monoliths allow penetration of large pDNA molecules to the internal surface area at high flow rate with low pressure drop. Different types of monolithic supports are currently available based on preparation and chemistry. These are silica-based, polyacrylamide-based and gel-based monolithic resins. Polymethacrylate monolithic support is an optimal adsorbent for pDNA separation. These adsorbents have large pore diameters and thus no significant hindrance to convective mass transport. They are resistant to pH, non-toxic, economically favorable to synthesize and can be easily modified by functionalizing with anion-exchange, hydrophobic interaction or affinity ligand. The flexibility and the ease to tailor their pore and surface characteristics to the target pDNA molecule through alteration in synthesis conditions make them more attractive.

In this current study, different standard media (LB, TB and SOC) for culturing recombinant E. coli DH5alpha harboring pUC19 were compared to a medium optimized for pDNA production. Lab scale, shake flask fermentations using the standard media showed that the highest pDNA volumetric and specific yields were for TB (11.4 mg/l and 6.3 mg/g dry cell mass respectively) and the lowest was for LB (2.8 mg/l and 3.3 mg/g dry cell mass respectively). A fourth medium, PDMR, designed by modifying a stoichiometrically-formulated medium with an optimized carbon source concentration and carbon to nitrogen ratio displayed pDNA volumetric and specific yields of 23.8 mg/l and 11.2 mg/g dry cell mass respectively. However, it is the economic advantage of the optimized medium that makes it so attractive. Keeping all variables constant except medium and using LB as a base scenario (100 MC units / mg pDNA), the optimized 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 optimized for pDNA production. Results for batch and fed batch fermentation employing PDMR and economic discussion of medium selection for large scale production of pDNA will be presented.

A monolithic sorbent was synthesized via free radical copolymerization of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with pore diameter tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with 2-Chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) for anion-exchange direct isolation of pDNA from clarified lysate obtained from E. coli DH5alpha-pUC19 culture in a ribonuclease/protease-free environment. Characterization of the monolithic resin showed a porous material with 68 % of the pores existing in the matrix having diameters above 300 nm. The final product isolated from a single-stage 5 minutes anion-exchange purification was a pure and homogenous SCpDNA with no endotoxins, gDNA, RNA and protein contaminations as confirmed with EtBr-AGE, enzyme restriction analysis, SDS-PAGE and limulus amoebocyte lysate. This cost effective and non-toxic technique is cGMP compatible and highly scalable for commercially-viable rapid production of pDNA.