Industry Overview

Plasmid technologies such as DNA vaccines are unique in the sense that they are one of the few technologies that can be created based on rational design1. This enables them to bypass a great deal of the research and development stages, so they can start being tested during clinical trials for safety and efficacy etc. This is extremely important during epidemics when new vaccines must be developed and mass produced within a few weeks to meet public demand. Existing technologies would require half a year to produce vaccines at this scale and would employ technology and infrastructure that is immensely expensive, which drives up the cost to the consumer and makes it available only to a small minority of countries.

The reason plasmid technology is so vital is that, according to Carnes and Williams (2007) it has the “potential to be the most rapidly deployed vaccine platform for pandemic application.” Another setback about existing technologies is that they won´t allow the production of the 0.5- 4 mg doses of plasmid required for DNA vaccinations. To develop a manufacturing process two criteria must be met:

1. The fermentation yield as well as quality must be appropriate.
2. 2. Plasmid composition and final product purity must meet current standards set by regulatory bodies such as the EMA (European Medicines Agency) or FDA (Food and Drug Administration, USA).

Plasmid DNA (pDNA) used in the production of DNA vaccines are obtained using recombinant DNA2 produced using Escherichia coli (E.coli) as an expression vector3 (see Protein Expression systems). The three most critical unit operations required in plasmid processing are:

• Fermentation
• Cell Disruption
• Downstream Purification

Fermentation

The main aim of fermentation is to optimise the volumetric and specific yield of supercoiled plasmid. This former enables more cost effective, smaller scale fermentations while the latter allows for greater plasmid purity. This is beneficial for the downstream processing steps as it enables fewer purification steps, curbing processing time. Also, reducing the volume of the fermentation paste is preferable because large amounts of fermentation paste can´t be processed for cell lysis procedures, this side steps a major bottleneck. Fermentation plays a major role in plasmid quality. It is during this stage that we ensure proper optimisation of quality plasmid i.e. plasmids in their supercoiled form which the FDA recognises to be more effective than plasmids in nicked and/or open circular forms.

Cell Disruption

After we grow the engineered cells to have the desired target plasmids, in order to extract the plasmids from the cell we must break open the cells. There are two kinds of methods for doing this: physio-mechanical and chemical methods. These steps must be done while inflicting minimal damage on the desired pDNA product caused by shear stress4 to the cell. Cell disruption must also be done such that there is minimal release of contaminants from the cell which may degrade and/or contaminate the pDNA and causes problems when separating the product from the lysate5 during downstream processing. Another reason existing cell disruption methods are unfavourable is that it requires vast amounts of harsh chemicals which result in large waste streams that are an environmental hazard.

Downstream Purification

Downstream processing aims to separate the product from the remaining lysate and is currently the most expensive part of the production process. The reason it´s so expensive is that the desired product takes up a small percentage of the total fermentation broth and hence, the lysate. Separation of pDNA from the remaining lysate is especially difficult since desired pDNA is difficult to differentiate from nucleic acid dispersed throughout the lysate.